17 beta-Hydroxysteroid dehydrogenase activity towards estradiol-17 beta has been demonstrated in the 105,000 x g supernatant of rabbit uterus. Hydroxylapatite chromatography of the enzyme activity isolated by ammonium sulfate precipitation, gel filtration and DEAE-cellulose chromatography yielded a single 17 beta-hydroxysteroid dehydrogenase activity. Further purification of the enzyme preparation by isoelectric focusing resulted in multiple peaks of activity. The molecular weight of the enzyme, caculated from mobility data on Sephadex gel, is approximately 64,000. Some properties of partially purified 17 beta-hydroxysteroid dehydrogenase activity have been studied. Estradiol-17 beta reacts at a faster rate than testosterone. The Km for estradiol is 4.16 x 10(-5) mol/l for the NAD-linked enzyme activity and 4.37 x 10(-5) mol/l when NADP as cofactor was used. The ratio of the maximal velocity for NADP to that for NAD was 1.42. The pH-optimum for estradiol appears between 9.5 and 10.5 and for estrone between 5.5 and 6.5. The enzyme appears to be of the sulfhydryl type.