The influence of various estrogens, progestogens and of cyanoketone (2 alpha-cyano-4,4,17 alpha-trimethylandrost-5-en-17 beta-ol-3-one) on the enzyme activity of delta 5-3 beta-hydroxysteroid dehydrogenase (HSD) (EC 1.1.1.145) was studied. Extracts of Pseudomonas testosteroni, rat testis total homogenate and a microsomal preparation were used as enzyme sources. Spectrophotometric determinations and the conversion of 3H-labelled dehydroepiandrosterone to androstenedione were used to assay for enzyme activities. Michaelis constants for dehydroepiandrosterone as substrate were 1.0 X 10(-5) mol, 1.5 X 10(-5) mol and 5 X 10(-6) mol for the bacterial enzyme, total homogenate and microsomal rat testis preparation, respectively. Dixon plot analysis was used to calculate apparent inhibition constants. Differences in the inhibition by steroids of the bacterial and testicular enzyme preparation were noted. The bacterial enzyme was inhibited by norethisterone (Ki = = 13.7 X 10(-6) mol) and by norethisterone acetate (Ki = 12.1 X 10(-6) mol), whereas no inhibition by norethisterone was noted in the rat testis enzyme assay systems. Norethisterone acetate was found to compete very weakly (Ki = 150 X 10(-6) mol) for binding to the active site of the rat testis microsomal enzyme. In addition, ethinylestradiol had an apparent inhibition constant of one tenth of the Michaelis constant in all enzyme preparations. The present data combine to suggest differences in the binding affinities of the catalytic sites between bacterial and testicular HSD systems. In addition, results of this investigation suggest that neither norethisterone nor a combination of norethisterone and ethinylestradiol cause hypospadias by inhibition of HSD activity in fetuses, whose mothers were treated with these steroids.