Tibolone [[7alpha,17alpha]-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one] is used to treat climacteric symptoms and prevent osteoporosis. It exerts tissue-selective effects via site-specific metabolism into 3alpha- and 3beta-hydroxymetabolites and a Delta4-isomer. Recombinant human cytosolic aldo-keto reductases 1C1 and 1C2 (AKR1C1 and AKR1C2) produce 3beta-hydroxytibolone, and the liver-specific AKR1C4 produces predominantly 3alpha-hydroxytibolone. These observations may account for the appearance of 3beta-hydroxytibolone in target tissues and 3alpha-hydroxytibolone in the circulation. Using liver autopsy samples (which express AKR1C1-AKR1C4), tibolone was reduced via 3alpha- and 3beta-hydroxysteroid dehydrogenase (HSD) activity. 3beta-Hydroxytibolone was exclusively formed in the cytosol and was inhibited by the AKR1C2-specific inhibitor 5beta-cholanic acid-3alpha, 7alpha-diol. The cytosolic formation of 3alpha-hydroxytibolone was inhibited by an AKR1C4-selective inhibitor, phenolphthalein. The ratio of these stereoisomers was 4:1 in favor of 3beta-hydroxytibolone. In HepG2 cell cytosol and intact cells (which do not express AKR1C4), tibolone was exclusively reduced to 3beta-hydroxytibolone and was blocked by the AKR1C1-AKR1C3 inhibitor flufenamic acid. In primary hepatocytes (which express AKR1C1-AKR1C4), time-dependent reduction of tibolone into 3beta- and 3alpha-hydroxytibolone was observed again in a 4:1 ratio. 3beta-HSD activity was inhibited by both 5beta-cholanic acid-3alpha,7alpha-diol and flufenamic acid, implicating a role for AKR1C2 and AKR1C1. By contrast, the formation of 3alpha-hydroxytibolone was exclusively inhibited by phenolphthalein implicating AKR1C4 in this reaction. 3beta- and 3alpha-Hydroxytibolone were rapidly metabolized into polar metabolites (>85%). The formation of minor amounts of tibolone was also observed followed by AKR1C-catalyzed epimerization. The low hepatic formation of 3alpha-hydroxytibolone suggests that AKR1C4 is not the primary source of this metabolite and instead it maybe formed by an intestinal or enterobacterial 3alpha-HSD.