A major and a minor form of dihydrodiol dehydrogenase were co-purified with 17 beta-hydroxysteroid dehydrogenase and aldehyde reductase, respectively, to apparent homogeneity from liver cytosol of male ddY mice. The activities of dihydrodiol dehydrogenase and testosterone dehydrogenase or aldehyde reductase of the two enzyme forms comigrated electrophoretically. The major form of the enzyme oxidized 17 beta-hydroxysteroids and nonsteroidal alicyclic alcohols and reduced 17-ketosteroids and various synthetic carbonyl compounds, showing higher affinity for steroids than for xenobiotics. The activity of this enzyme form toward benzene dihydrodiol and testosterone exhibited identical thermostability and susceptibility to inhibition by quercitrin, SH-reagents, nonsteroidal estrogens and anti-inflammatory agents. On the other hand, the minor form of the enzyme, which oxidized benzene dihydrodiol but not 17 beta-hydroxysteroids, also reduced various aldehydes well and was specifically inhibited by barbiturates and sorbinil. These results indicate that the major form of dihydrodiol dehydrogenase is identical to 17 beta-hydroxysteroid dehydrogenase and the minor enzyme form to aldehyde reductase.