A procedure for isolation of a highly-purified estrophilic hydroxysteroid dehydrogenase (EHSD) from rabbit liver, including ammonium sulphate fractionation, gel filtration, ion-exchange and affinity chromatography on estradiol-Sepharose, has been developed. The enzyme possesses NADP-dependent 3 alpha,3 beta,17 beta,20 alpha-HSD activities with a wide spectrum of androgenic, progestagenic, and estrogenic substrates. EHSD is a monomeric protein whose molecular mass determined by different methods is 35,000-39,000. The protein exhibits microheterogeneity due to the differences in molecular surface charge. The catalytic and hormone-binding properties and molecular sizes of the two protein fractions obtained by chromatography on DEAE-Toyopearl are close or identical. The enzymatic activity of EHSD is minor as compared to other HSDs from rabbit liver. However, the low values of Km, the high affinity for steroid ligands, and high tissue levels of EHSD suggest the protein to play a role in the biodynamics of sex hormones.