Ketoconazole (K) is an antifungal imidazole derivative which is a potent inhibitor of steroid biosynthesis in rodents and humans. To study the effect of K on rat ovarian steroidogenesis we measured the activities of five ovarian microsomal steroidogenic enzymes in K-treated rats and controls. Thirty hypophysectomized, gonadotropin-treated female adult rats were given either 2 mg K or water every 12 hours by mouth during 5 days. Mean ovarian weight was similar in both groups of animals. The K-treated group had an estradiol (E) serum concentration of 176 +/- 48 pg/ml whereas it was 278 +/- 56 pg/ml in the control group (NS). The K-treated animals had decreased activities of the 17,20-desmolase, 17-ketosteroid-reductase and aromatase enzymes. The 3 beta hydroxysteroid-dehydrogenase and 17-hydroxylase activities were similar in both groups. We conclude that K directly inhibits the activities of the 17,20-desmolase, 17-ketosteroid-reductase and aromatase enzymes in the rat ovary.