The enzyme was purified from the dialyzed preparation by removal of the precipitate formed at pH 6.2, and alumina C [alpha] [sic] treatment at pH 7.4. Both diphospho- and triphosphopyridine nucleotides were required as coenzymes. Optimum pH was 7.4-8.5 with the use of 7-[alpha]-hydroxycholesterol as a substrate. The activity was strongly inhibited by heavy metal ions or monoiodoacetate. The enzyme was active towards 3-[beta]-hydroxy- [DELTA] 5-cholenate, [DELTA] 4 androstene-3-[beta]-17-[beta]-diol, testisterone, 7-[alpha]-hydroxycholesterol, coprostanol, cholesterol, 7-oxocholesterol, and cholestanol. The enzyme catalyzes the dehydrogenation of 3-[beta]-hydroxy-45- sterols to corresponding 3-keto-4 4- sterols. The rate of 44-3-keto group formation determined by the change of E240 mu paralleled the amount of diphosphopyridine nucleotide that disappeared.