Multiple forms of the soluble 17 beta-hydroxysteroid dehydrogenase of female rabbit liver were identified. NAD-dependent and NADP-dependent enzyme activities were separated by affinity chromatography on agarose-immobilized Procion Red HE3B, and three forms of the NADP-dependent enzyme activity were purified by chromatofocusing. These three enzyme forms are charge isomers and have no quaternary structure. The enzymes catalysed the C-17 oxidoreduction of oestrogens and androgens; with all enzyme forms the activity towards androgens was higher than that toward oestrogens. The enzymes also exhibited 3 alpha-hydroxysteroid dehydrogenase activity towards androgens of the 5 beta-androstane series. Comparison of the relative activities of the enzymes towards a number of oestrogen and androgen substrates revealed differences among the enzyme forms for both the oxidative and the reductive reactions. In particular, one enzyme form had a significantly lower Km for the 3 alpha-hydroxysteroid substrate and a higher 3 alpha-/17 beta-hydroxysteroid dehydrogenase activity ratio than the other two enzyme forms.