The mechanism of reduction of cholesterol to coprostanol by Eubacterium coprostanoligenes ATCC 51222 was studied by incubating the bacterium with a mixture of alpha- and beta-isomers of [4-3H,4-14C]cholesterol. Coprostanol, isolated after incubation of [4-3H,4-14C]cholesterol in a growth medium under anaerobic conditions, retained 97% of the tritium originally present in cholesterol. The majority of this tritium (64%), however, was in the C-6 position in coprostanol, which showed that the conversion of cholesterol into coprostanol by E. coprostanoligenes involved the intermediate formation of 4-cholesten-3-one followed by the reduction of the latter to coprostanol. In resting cell assays in which washed bacterial cells were incubated with micellar cholesterol in phosphate buffer at 37 degrees C, both 4-cholesten-3-one and coprostanone were produced in addition to coprostanol. Furthermore, 5-cholesten-3-one, 4-cholesten-3-one, and coprostanone were converted efficiently to coprostanol by E. coprostanoligenes. These results support the hypothesis that the major pathway for reduction of cholesterol by E. coprostanoligenes involves the intermediate formation of 4-cholesten-3-one followed by reduction of the latter to coprostanol through coprostanone as an intermediate.