Homogeneous indanol dehydrogenase from monkey liver catalyzed the reversible conversion of 3 alpha- or 20 alpha-hydroxy groups of several bile acids and 5 beta-pregnanes to the corresponding 3- or 20-ketosteroids. The kcat values for the steroids determined at pH 7.4 were low, but the kcat/Km values for the 3-ketosteroids were comparable to or exceeded those for 1-indanol and xenobiotic carbonyl substrates. The enzyme transferred the 4-pro-R-hydrogen atom of NADPH to the 3 beta- or 20 beta-face of the ketosteroid substrate. Competitive inhibition of the hydroxysteroid dehydrogenase activity of the enzyme by medroxyprogesterone acetate, hexestrol, and 1,10-phenanthroline suggests that both 1-indanol and hydroxysteroid are oxidized at the same active site on the enzyme. The specific inhibitor of the enzyme, 1,10-phenanthroline, suppressed the 3 alpha-hydroxysteroid dehydrogenase activity in the crude extract of monkey liver by 50%. The results strongly suggest that indanol dehydrogenase acts as a 3(20)alpha-hydroxysteroid dehydrogenase in the metabolism of certain steroid hormones and bile acids.