He accepted a research post in the Department of Biochemistry, Cambridge, in 1942, and received the degree of Ph.D. in early 1951 for work on the mode of action of penicillin. In 1955 he was invited by Professor Michael Swann to set up a biochemical research unit, called the Chemical Biology Unit, in the Department of Zoology, Edinburgh University, where he was appointed to a Senior Lectureship in 1961, to a Readership in 1962, although ill health led to his resignation in 1963.
In the 1960s, ATP was known to be the energy currency of life, but the mechanism by which ATP was created in the mitochondria was assumed to be by substrate-level phosphorylation. Mitchell's chemiosmotic hypothesis was the basis for understanding the actual process of oxidative phosphorylation. At the time, the biochemical mechanism of ATP synthesis by oxidative phosphorylation was unknown.
Mitchell realised that the movement of ions across an electrochemical membrane potential could provide the energy needed to produce ATP. His hypothesis was derived from information that was well known in the 1960s. He knew that living cells had a membrane potential; interior negative to the environment. The movement of charged ions across a membrane is thus affected by the electrical forces (the attraction of plus to minus charges). Their movement is also affected by thermodynamic forces, the tendency of substances to diffuse from regions of higher concentration. He went on to prove that ATP synthesis was coupled to this electrochemical gradient.
His theory was confirmed by the discovery of ATP synthase, a membrane-bound protein that uses the potential energy of the electrochemical gradient to make ATP.