A mammalian hydroxysteroid dehydrogenase having both 3 alpha- and 17 alpha-enzyme activity is described for the first time. Multiple forms of this 3(17) alpha-hydroxysteroid dehydrogenase are present in the cytosol of female rabbit kidney. Four forms of this enzyme were resolved by a purification procedure which included DEAE-cellulose chromatography and isoelectric focusing and three of the isolated enzymes were homogeneous on the basis of polyacrylamide gel electrophoresis. The purified enzymes exhibited 17 alpha-enzyme activity toward both androgen and estrogen substrates and 3 alpha-enzyme activity toward androgens of the 5 alpha-androstane series. In general, the 3 alpha-enzyme activity was greater than the 17 alpha-enzyme activity and the latter activity was greater toward androgens than estrogens. There were differences in substrate specificity among the enzyme forms. In particular, with estrogen substrates one of the forms displayed a high specificity toward 17 alpha-estradiol 3-glucuronide.