The purified 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat liver cytosol is potently inhibited by the nonsteroidal anti-inflammatory drugs in rank-order of their therapeutic potency, i.e. by micromolar concentrations that would inhibit cyclooxygenase [T. M. Penning, and P. Talalay, Proc. natn. Acad. Sci. U.S.A. 80, 4504 (1983)]. In the present study, indomethacin-sensitive 3 alpha-hydroxysteroid dehydrogenase is shown to exist in seven rat tissues, including those that require androgens for growth (e.g. prostate) and those that rapidly metabolize prostaglandins (e.g. lung). Thus, the reduction of 5 alpha-dihydrotestosterone catalyzed by prostatic cytosol was potently inhibited by indomethacin (IC50 = 10 microM), while the reduction of 5 beta-dihydrocortisone catalyzed by liver, lung and testis was more sensitive to inhibition by this drug (IC50 1-3 microM). These data suggest that, under conditions in which cyclooxygenase is inhibited, androgen and cortisone metabolism may be affected. A surprising feature is that the specific activity of the indomethacin-sensitive dehydrogenase was higher in the lung than in tissues that are hormonally responsive (e.g. prostate and testis).