Homogeneous indanol dehydrogenase from monkey liver catalyzed the reversible conver-sion of 3α- or 20α-hydroxy groups of several bile acids and 5β-pregnanes to the corre-sponding 3- or 20-ketosteroids. The k_cat values for the steroids determined at pH7.4 werelow, but the k_cat/K_m values for the 3-ketosteroids were comparable to or exceeded those for1-indanol and xenobiotic carbonyl substrates. The enzyme transferred the 4-pro-R-hydrogen atom of NADPH to the 3β- or 20β-face of the ketosteroid substrate. Competitiveinhibition of the hydroxysteroid dehydrogenase activity of the enzyme by medroxyproges-terone acetate, hexestrol, and 1, 10-phenanthroline suggests that both 1-indanol andhydroxysteroid are oxidized at the same active site on the enzyme. The specific inhibitor ofthe enzyme, 1, 10-phenanthroline, suppressed the 3α-hydroxysteroid dehydrogenase activ-ity in the crude extract of monkey liver by 50%. The results strongly suggest that indanoldehydrogenase acts as a 3 (20)α-hydroxysteroid dehydrogenase in the metabolism of certainsteroid hormones and bile acids.