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Urey, born the son of a teacher and lay minister in Walkerton, Indiana, was educated at the universities of Montana, where he studied zoology, and California, where he obtained a PhD in chemistry (1923). After a year at the Institute of Theoretical Physics in Copenhagen, he began his teaching career at Johns Hopkins in 1924. In 1929 he moved to Columbia University, remaining there until 1958 when he became a professor at the University of California.

He is best known as the discoverer of deuterium – the isotope of hydrogen containing one proton and one neutron in its nucleus. This followed the accurate measurement of the atomic weights of hydrogen and oxygen by Francis W. Aston and the discovery of oxygen isotopes by William Giauque.

To obtain deuterium Urey used the fact that it would evaporate at a slightly slower rate than normal hydrogen. He took some four liters of liquid hydrogen, which he distilled down to a volume of one cubic centimeter. The presence of deuterium was then proved spectroscopically. Urey went on to investigate differences in chemical-reaction rate between isotopes. During World War II he was in charge of the separation of isotopes in the atomic-bomb project. Urey's research also led to a large-scale method of obtaining deuterium oxide (heavy water) for use as a neutron moderator in reactors.

His interest in isotope effects in chemical reactions gave him the idea for a method of measuring temperatures in the oceans in the past. It depended on the fact that the calcium carbonate in shells contains slightly more oxygen–18 than oxygen–16, and the ratio depends on the temperature at which the shell formed.

For his discovery of heavy hydrogen Urey received the 1939 Nobel Prize for chemistry.