In an in vitro system, ketoconazole inhibited the aromatization of [4-14C]-testosterone to estradiol-17 beta by rat ovarian tissue, in a dose-related manner. The amount of an intermediate, 4-androsten-17 beta, 19-diol-3-one, formed was also reduced. When the latter steroid or a subsequent intermediate, 4-androsten-19-aldehyde-3, 17-dione, was used as substrate, ketoconazole at approximately three times the molar concentration of the substrate, depressed estradiol-17 beta synthesis to 38% and 66% of control respectively. With ketoconazole in a concentration of about thirty times that of the substrate, estradiol-17 beta synthesis was depressed to approximately 10% that of control. It is concluded that ketoconazole inhibits all three sequential hydroxylation steps leading to the ovarian aromatization of androgens.