We previously reported that the human Y' bile acid binder, which has higher bile acid binding affinities than rat Y' binders (3a-hydroxysteroid dehydrogenases), has dihydrodiol dehydrogenase activity and is different from 3a-hydroxyeteroid dehydrogenases. In this study, 3a-hydmxyeteroid dehydrogenases and 38hydroxysteroid dehydrogenase were purified from human liver, and bile acid binding affinities and enzyme kinetics of the 3a-hydroxysteroid dehydrogenases were studied. On chromatofocusing of pooled Affigel blue fraction of the Y' fraction, three hhydmxyeteroid dehydrogenase peaks eluted at pH 6.0,6.7 and 5.4. These peaks did not bind bile acids, and further purification by hydroxyapatite-highperformance liquid chromatography gave pure 3ahydroxysteroid dehydrogenases with identical M, (36,OOO) having dihydrodiol dehydrogenase activity. 3g-Hydroqsteroid dehydrogenase was eluted together with Y' bile acid binder at pH 7.2 on chromatofocusing and was separated from Y' bile acid binder on hydroxyapatite-high-performance liquid chromatography as a pure protein with M, 32,000. The apparent &s of 3u-hydmxyeteroid dehydrogenases were similar to those of rat enzymes. In conclusion, we purified human hepatic hhydroxysteroid dehydrogenases, which have similar characteristics to rat enzymes, but do not bind bile acids or reduce bile acid precursors. These data further support the importance of human bile acid binder in intracellular bile acid transport in the human liver. (HEPATOLOGY 1992;16:366-371.) Three different classes of proteins bind bile acids with high a n i t i e s in rat and human liver cytosol: Y protein or glutathione S-transferases (1-4); Y' protein (1, 4, 5) and Z protein or fatty acid binding protein (6). Among these proteins, rat Y' bile acid binders are identical to 3a-hydroxysteroid dehydrogenases (3a-HSD) (EC 1.1.1.501, which catalyze the oxidoreduction of C,position of bile acids (7). Furthermore, we have reported that bile acids interact with Y' bile acid binders in intact rat hepatocytes using isolated hepatocytes and single-pass perfused liver (8, 9). Under physiological ~~~ ~