A homogenate of cells of Streptomyces hydrogenans which have been cultivated in the presence of estradiol-17beta, was separated by gel filtration on Sephadex G-200. In the presence of NADH, a few distinct fractions converted about 10% of [3H]toesterone to 5alpha-dihydrotesterone (5alpha-DHT), 5alpha-androstanedione, 4-androstenedione, and dehydroepiandrosterone (DHEA). The metabolites were separated after two consecutive runs on silica gel and propanediol-1,2-impregnated cellulose plates by thin layer chromatography. Identification was achieved by comparison with known steroid samples, specific staining procedures and by crystallization to constant specific radioactivity. The 5alpha-reductase activity responsible for the formation of 5alpha-DHT and 5alpha-androstanedione in vitro required NADH as co-substrate and could only be found after induction of the cells with estradiol-17beta in vivo. Within the same chromatographic fractions, a delta4-delta5-ketosteroid isomerase activity can be detected which catalyzes the reverse reaction from the 4-androstene testosterone to the 5-anthrostene DHEA in vitro.