The enzyme converting 5-cholestene-3 beta, 7 alpha-diol to 7 alpha-hydroxy-4-cholesten-3-one has been solubilized from rabbit liver microsomes by treatment with a mixture of sodium cholate and the nonionic detergent Renex 690. The enzyme was purified about 200-fold, with a recovery of more than 50%, by chromatography on DEAE-cellulose, hydroxylapetite, 2',5',ADP-Sepharose 4B and 5'-AMP-Sepharose 4B. The purified enzyme showed only one protein band, with an apparent molecular weight of 46,000, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was eluted as a single peak on gel filtration on Ultrogel AcA 34. The elution volume corresponded to that observed for globular proteins with molecular weights in the 45,000 to 50,000 region. The substrate specificity of the microsomal fraction and of the purified oxidoreductase in oxidation and reduction of various 3-oxygenated C19-, C21-, and C27-steroids was studied in the presence of NAD and NADH. Whereas the microsomal fraction had a broad substrate specificity, NAD-supported oxidation with the purified oxidoreductase only occurred with 5-cholestene-3 beta, 7 alpha-diol as substrate. NADP could not replace NAD in the reaction. NADH-supported reduction with the purified oxidoreductase only occurred with 7 alpha-hydroxy-5 alpha-cholestan-3-one as substrate. The results suggest that conversion of 5-cholestene-3 beta, 7 alpha-diol to 7 alpha-hydroxy-4-cholesten-3-one is catalyzed by a single enzyme specific for certain C27-steroids.