The effect of cyanoketone on some steps of steroidogenesis has been studied on testicular and adrenal tissue of male Wistar rats. Pregnenolone, dehydroepiandrosterone and progesterone (4-C14) have been used as substrates. The metabolites have been separated by thin layer chromatography and identified by crystallization to constant specific activity. The results have shown that cyanoketone inhibits the activity of 3β-hydroxysteroid dehydrogenase; 17α-hydroxylase is not inhibited in adrenal tissue, while in testicular tissue the metabolic pathway pregnenolone →17α-hydroxypregnenolone – > dehydroepiandrosterone is altered and testosterone biosynthesis is almost completely inhibited.