The pregnene derivative, 4-pregnene-3-one-20 beta-carboxaldehyde (22-A) was evaluated as an inhibitor of 17 alpha-hydroxylase/C17,20-lyase in rat testicular microsomes and of 5 alpha-reductase in human prostatic homogenates. The effect of the compound in vivo was studied in adult male rats. The 22-A demonstrated potent and competitive inhibition of 17 alpha-hydroxylase and C17,20-lyase with Ki values 8.48 and 0.41 microM, respectively, significantly below the Km values for these two enzymes (33.75 and 4.55 microM). This compound also showed potent inhibition of 5 alpha-reductase with a Ki value of 15.6 nM (Km for this enzyme is 50 nM). By comparison, ketoconazole, a currently studied 17 alpha-hydroxylase/C17,20-lyase inhibitor for the treatment of prostatic cancer, showed less potent inhibition of 17 alpha-hydroxylase (Ki 39.5 microM) and C17,20-lyase (Ki 3.6 microM) and did not inhibit 5 alpha-reductase. Progesterone which has been reported to inhibit the 17 alpha-hydroxylase/C17,20-lyase, did not significantly reduce the production of testosterone by rat testes in vitro in comparison to controls, while the same concentration of 22-A demonstrated a 42% reduction of testosterone biosynthesis. When the adult male rats were injected s.c. with 22-A at 50 mg/day/kg for a 2 week period, the testosterone concentrations in the rat sera were significantly lower than control values (P less than 0.05), whereas serum corticosterone levels did not change. These results suggest that 22-A is a selective potent inhibitor for 17 alpha-hydroxylase and C17,20-lyase, but is more potent for the C17,20-lyase. The compound also inhibits 5 alpha-reductase, and therefore may reduce biosynthesis of testosterone and dihydrotestosterone effectively. Thus, 22-A may be useful in the treatment of problems associated with the androgen excess and prostatic cancer.