The 20 alpha-hydroxysteroid dehydrogenase activity in human term placenta has been localized by different investigators to nuclear, mitochondrial, microsomal, and cytosolic subcellular fractions. Furthermore, in the cytosol, 20 alpha-hydroxysteroid dehydrogenase activity may be a second function of the enzyme that mediates 17 beta-estradiol dehydrogenase activity. To search for a unique 20 alpha-hydroxysteroid dehydrogenase, human placental villous tissue, homogenized in three different buffer systems, was fractionated by differential centrifugation, and the 17 beta- and 20 alpha-activities were measured by radioisotope conversion assay. The enrichment and purity of the subcellular fractions were shown by marker enzyme assays and electron microscopy studies. Under all experimental conditions, 20 alpha-hydroxysteroid dehydrogenase activity was identified only in the 105,000 g placental cytosol: intact, osmotically ruptured, and acetone-extracted mitochondria, nuclei, and microsomes did not convert progesterone to 20 alpha-dihydroprogesterone. Furthermore, because 17 beta-estradiol dehydrogenase activity was in large part soluble in the cytosol, these localization studies are consistent with the hypothesis that the 20 alpha- and 17 beta-oxidoreductase activities in human placenta reside on one soluble protein.