Cholesterol oxidase synthesis ability by various fast-growing mycobacteria (M. fortuitum, M. vaccae, M. phlei, M. smegmatis) and Rhodococcus sp. IM 58 was investigated by thin-layer chromatography and Western blot analysis. In contrast with the case of cholesterol oxidases from Arthrobacter sp. IM 79 and Streptomyces sp., those from the strains tested were localized intracellularly. Western blot experiments revealed high antigenic similarity of those enzymatic proteins to cholesterol oxidase from Schizophyllum commune, and lack of homology to cholesterol oxidase from Streptomyces sp. Differences in the analyzed enzymes were due not only to a single antigenic determinant alteration but also to the significant distinctions on the genetic level as shown by Southern blot hybridization.