The cytosol 3alpha-hydroxysteroid dehydrogenase of rat ventral prostate has been partially purified. The rates of both the oxidation and reduction by crude and partially purified enzymes have been measured with a variety of radioactive substrates, and the effects of several inhibitor steroids have been assessed. Four conclusions have been drawn from the study. First, no detectable 3beta-androstanediol was formed from dihydrotestosterone, and the oxidation of 3beta-androstanediol was undetectable. Second, the cytosol enzyme exhibits a distinct and unique substrate specificity in that steroids with keto or hydroxyl substitution on the 11th carbon of the steroid cannot serve as substrates or as inhibitors of the enzyme. Third, either 5alpha or 5 beta reduction of delta4,3-keto steroids must take place before the steroids can serve as substrates of the enzyme. Fourth, many delta4,3-keto steroids that cannot act as substrates for the enzyme inhibit the enzyme competitively and may well serve as physiological regulators of the reaction in intact cell.