Estradiol 17 alpha-dehydrogenase (EC 1.1.1.148) and estradiol 17 beta-dehydrogenase (EC 1.1.1.62) from horse placenta have been purified to homogeneity. Both enzymes are localized in the microsomal fraction and are solubilized in 1.5% sodium cholate. The 17 alpha- and 17 beta-dehydrogenases are separated by selective elution from hydroxylapatite with 0.5 and 1.0 M potassium phosphate, respectively. Subsequent purification is achieved by two affinity-absorption steps using reactive blue 2-agarose and estriol hemisuccinate-Sepharose. Homogeneous estradiol 17 alpha-dehydrogenase has a specific activity of 10 IU/mg and has been purified 5900-fold with an 87% recovery. Homogeneous estradiol 17 beta-dehydrogenase has a specific activity of 10.6 IU/mg and has been purified 15 000-fold with an apparent recovery of 100%. Each enzyme exhibits a single band on polyacrylamide disc gel electrophoresis and on sodium dodecyl sulfate (SDS) gel electrophoresis. The mobilities of the two on SDS gels are identical and correspond to subunit molecular weights of 33000. The apparent molecular weight of the undenatured, active enzyme, as determined by gel filtration on Sephacryl S-300, is 52000 in the case of the 17 alpha-dehydrogenase and 68500 for the 17 beta-dehydrogenase. Both enzymes exhibit pH optima at 9.0-9.5; both prefer NAD+ over NADP+ but utilize both cofactors. Both are highly specific for their respective epimers of estradiol with apparent Km values of 1.7 microM at pH 9.5.