Rat liver cytosol was previously shown to contain at least three NADPH-dependent enzymes which catalyze the hydrogenation of chloral hydrate. Two of them had previously been shown to catalyze the hydrogenation of long-chain aliphatic and aromatic aldehydes or aromatic ketones in addition to halogenated acetaldehydes. The present paper demonstrates that one of these enzymes also catalyzes the reversible hydrogenation of 3-oxosteroids to 3 alpha-hydroxysteroids. We confirmed that the enzyme catalyzes the reversible hydrogenation of C23, C24 and C26 3-oxo bile acids with a 1:1 stoichiometry. The substitution at C-12 with a hydroxyl group markedly decreased the reaction rate, and the rate of dehydrogenation of C20, C23, C26, C24, and C25 bile acids decreased in the descending order. In general, the conjugation with glycine or taurine increased the reaction rate, while the replacement of the terminal carboxyl group of the C24 compound with an isobutyl group markedly decreased it. 3 alpha-Hydroxy bile acids with A/B trans configuration were also preferable substrates for the enzyme, although the reaction rate was relatively low compared with that of those with A/B cis configuration. The enzyme may be involved in the biosynthesis of lithocholic acid from cholesterol through 3 alpha-hydroxy-5-cholen-24-oic acid, which was proposed by Mitropoulos and Myant (Mitropoulos & Myant (1967), Biochem. J. 103, 472--479).