The extension of this idea to substances in general necessarily led him to the law of multiple proportions, and the comparison with experiment brilliantly confirmed his deduction. It may be noted that in a paper on the proportion of the gases or elastic fluids constituting the atmosphere, read by him in November 1802, the law of multiple proportions appears to be anticipated in the words: "The elements of oxygen may combine with a certain portion of nitrous gas or with twice that portion, but with no intermediate quantity", but there is reason to suspect that this sentence may have been added some time after the reading of the paper, which was not published until 1805. Compounds were listed as binary, ternary, quaternary, etc. (molecules composed of two, three, four, etc. atoms) in the New System of Chemical Philosophy depending on the number of atoms a compound had in its simplest, empirical form. He hypothesized the structure of compounds can be represented in whole number ratios. So, one atom of element X combining with one atom of element Y is a binary compound. Furthermore, one atom of element X combining with two elements of Y or vice versa, is a ternary compound. Many of the first compounds listed in the New System of Chemical Philosophy correspond to modern views, although many others do not. Dalton used his own symbols to visually represent the atomic structure of compounds. These have made it in New System of Chemical Philosophy where Dalton listed a number of elements, and common compounds.

• The atoms of a given element are different from those of any other element; the atoms of different elements can be distinguished from one another by their respective relative atomic weights.
• All atoms of a given element are identical.
• Atoms of one element can combine with atoms of other elements to form chemical compounds; a given compound always has the same relative numbers of types of atoms.
• Atoms cannot be created, divided into smaller particles, nor destroyed in the chemical process; a chemical reaction simply changes the way atoms are grouped together.
• Elements are made of tiny particles called atoms.

Dalton proposed an additional "rule of greatest simplicity" that created controversy, since it could not be independently confirmed.

When atoms combine in only one ratio, "..it must be presumed to be a binary one, unless some cause appear to the contrary".

This was merely an assumption, derived from faith in the simplicity of nature. No evidence was then available to scientists to deduce how many atoms of each element combine to form compound molecules. But this or some other such rule was absolutely necessary to any incipient theory, since one needed an assumed molecular formula in order to calculate relative atomic weights. In any case, Dalton's "rule of greatest simplicity" caused him to assume that the formula for water was OH and ammonia was NH, quite different from our modern understanding.

Despite the uncertainty at the heart of Dalton's atomic theory, the principles of the theory survived. To be sure, the conviction that atoms cannot be subdivided, created, or destroyed into smaller particles when they are combined, separated, or rearranged in chemical reactions is inconsistent with the existence of nuclear fusion and nuclear fission, but such processes are nuclear reactions and not chemical reactions. In addition, the idea that all atoms of a given element are identical in their physical and chemical properties is not precisely true, as we now know that different isotopes of an element have slightly varying weights. However, Dalton had created a theory of immense power and importance. Indeed, Dalton's innovation was fully as important for the future of the science as Antoine Laurent Lavoisier's oxygen-based chemistry had been.

Dalton communicated his atomic theory to Thomson who, by consent, included an outline of it in the third edition of his System of Chemistry (1807), and Dalton gave a further account of it in the first part of the first volume of his New System of Chemical Philosophy (1808). The second part of this volume appeared in 1810, but the first part of the second volume was not issued till 1827. This delay is not explained by any excess of care in preparation, for much of the matter was out of date and the appendix giving the author's latest views is the only portion of special interest. The second part of vol. ii. never appeared.

He was president of the Lit & Phil from 1817 until his death, contributing 116 memoirs. Of these the earlier are the most important. In one of them, read in 1814, he explains the principles of volumetric analysis, in which he was one of the earliest workers. In 1840 a paper on the phosphates and arsenates, often regarded as a weaker work, was refused by the Royal Society, and he was so incensed that he published it himself. He took the same course soon afterwards with four other papers, two of which (On the quantity of acids, bases and salts in different varieties of salts and On a new and easy method of analysing sugar) contain his discovery, regarded by him as second in importance only to the atomic theory, that certain anhydrates, when dissolved in water, cause no increase in its volume, his inference being that the salt enters into the pores of the water.

James Prescott Joule was a famous pupil of Dalton.

As an investigator, Dalton was often content with rough and inaccurate instruments, though better ones were obtainable. Sir Humphry Davy described him as "a very coarse experimenter", who almost always found the results he required, trusting to his head rather than his hands. On the other hand, historians who have replicated some of his crucial experiments have confirmed Dalton's skill and precision.

In the preface to the second part of Volume I of his New System, he says he had so often been misled by taking for granted the results of others that he determined to write "as little as possible but what I can attest by my own experience", but this independence he carried so far that it sometimes resembled lack of receptivity. Thus he distrusted, and probably never fully accepted, Gay-Lussac's conclusions as to the combining volumes of gases. He held unconventional views on chlorine. Even after its elementary character had been settled by Davy, he persisted in using the atomic weights he himself had adopted, even when they had been superseded by the more accurate determinations of other chemists. He always objected to the chemical notation devised by Jöns Jakob Berzelius, although most thought that it was much simpler and more convenient than his own cumbersome system of circular symbols.

Before he had propounded the atomic theory, he had already attained a considerable scientific reputation. In 1804, he was chosen to give a course of lectures on natural philosophy at the Royal Institution in London, where he delivered another course in 1809–1810. However, some witnesses reported that he was deficient in the qualities that make an attractive lecturer, being harsh and indistinct in voice, ineffective in the treatment of his subject, and singularly wanting in the language and power of illustration.

In 1810, Sir Humphry Davy asked him to offer himself as a candidate for the fellowship of the Royal Society, but Dalton declined, possibly for financial reasons. However, in 1822 he was proposed without his knowledge, and on election paid the usual fee. Six years previously he had been made a corresponding member of the French Académie des Sciences, and in 1830 he was elected as one of its eight foreign associates in place of Davy. In 1833, Earl Grey's government conferred on him a pension of £150, raised in 1836 to £300.

Dalton never married and had only a few close friends. He lived for more than a quarter of a century with his friend the Rev. W. Johns (1771–1845), in George Street, Manchester, where his daily round of laboratory work and tuition was broken only by annual excursions to the Lake District and occasional visits to London. In 1822 he paid a short visit to Paris, where he met many distinguished resident scientists. He attended several of the earlier meetings of the British Association at York, Oxford, Dublin and Bristol.

Dalton suffered a minor stroke in 1837, and a second one in 1838 left him with a speech impediment, though he remained able to do experiments. In May 1844 he had yet another stroke; on 26 July he recorded with trembling hand his last meteorological observation. On 27 July, in Manchester, Dalton fell from his bed and was found lifeless by his attendant. He was buried in Manchester in Ardwick cemetery. The cemetery is now a playing field, but pictures of the original grave are in published materials.

A bust of Dalton, by Chantrey, was publicly subscribed for and placed in the entrance hall of the Royal Manchester Institution. Chantrey also crafted a large statue of Dalton, now in the Manchester Town Hall. In honour of Dalton's work, many chemists and biochemists use the (as yet unofficial) unit dalton (abbreviated Da) to denote one atomic mass unit, or 1/12 the weight of a neutral atom of carbon-12. There is a John Dalton Street connecting Deansgate and Albert Square in the centre of Manchester. Manchester Metropolitan University has a building named after John Dalton and occupied by the Faculty of Technology, in which the majority of its Science & Engineering lectures and classes take place. A statue is outside the John Dalton Building of the Manchester Metropolitan University in Chester Street which has been moved from Piccadilly. It was the work of William Theed (after Chantrey) and is dated 1855 (it was in Piccadilly until 1966). The University of Manchester had a hall of residence called Dalton Hall; it also established two Dalton Chemical Scholarships, two Dalton Mathematical Scholarships, and a Dalton Prize for Natural History. There is also a Dalton Medal awarded occasionally by the Manchester Literary and Philosophical Society (only 12 times altogether). A lunar crater has been named after Dalton. "Daltonism" became a common term for colour blindness and "Daltonien" is the actual French word for "colour blind". The name Dalton can often be heard in the halls of many Quaker schools, for example, one of the school houses in Coram House, the primary sector of Ackworth School, is called Dalton.

Much of his collected work was damaged during the bombing of the Manchester Literary and Philosophical Society on 24 December 1940. This event prompted Isaac Asimov to say, "John Dalton's records, carefully preserved for a century, were destroyed during the World War II bombing of Manchester. It is not only the living who are killed in war." The damaged papers are now in the John Rylands Library having been deposited in the university library by the society.