We have demonstrated that the isomerization reaction catalyzed by Brevibacterium sterolicum (ATCC 81387) cholesterol oxidase (EC 1.1.3.6) proceeds via a stereospecific proton transfer from the 4 beta carbon to the 6 beta carbon to form 4-cholestene-3-one using deuterated and nondueterated substrates. This result implies that there is one active site base, positioned over the beta-face, responsible for isomerization. On the basis of X-ray crystallographic evidence [Li, J., Vrielink, A., Brick, P. & Blow, D. M. Biochemistry 32, 11507-11515 (1993)], glutamate-361 is the most likely candidate for this general base.