In 1939, Cain  joined Howard Florey to investigate natural antibacterial agents produced by microorganisms at Oxford University. This led him and Florey to revisit the work of Alexander Fleming, who had described penicillin earlier. Chain and Florey went on to discover penicillin's therapeutic action and its chemical composition. He also theorized the structure of penicillin, which was confirmed by X-ray crystallography done by Dorothy Hodgkin. For this research, Chain, Florey, and Fleming received the Nobel Prize for Physiology and Medicine in 1945.