Cholesterol oxidase produced by Rhodococcus equi MIL 1037 was purified from the supernatant solution of the culture broth by ammonium sulfate fractionation, DEAE- and CM-cellulose column chromatography, hydroxyapatite column chromatography and Sephadex G-75 gel filtration. The resulting enzyme was shown homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated as 48, 000 by SDS-gel electrophoresis. The enzyme contained 453 amino acid residues including 40 moles of alanine and 16 moles of tryptophan. The enzyme was specific to 3β-hydroxysteroids and its relative activity (RA) was 37. 5 for β-sitosterol, 25 for 5α-cholestan-3β-ol, 25 for pregnenolone and 0. 1 for stigmasterol on the basis of 100 for cholesterol. The Km value for the oxidation of cholesterol was 0.33 mM. The enzyme was inhibited by CuSO4, AgNO3 and HgCl2, and partially inhibited by p-chloromercuribenzoic acid, but unaffected by metal chelating reagents.