Rat ovarian 20 alpha-hydroxysteroid dehydrogenase was purified 230-fold with a 48% recovery through a 3-step process involving hydrophobic, gel filtration and green dye affinity chromatography. The purified enzyme was demonstrated to be a single polypeptide chain of Mr 36 000. Initial velocity studies of all four substrates in the forward and reverse reactions indicated a sequential mechanism for the enzyme. Product inhibition and dead-end inhibition studies with substrate analogs were consistent with an ordered bi-bi mechanism in which NADP is the first substrate bound to the enzyme and NADPH, the second product released. Several NADP analogs were demonstrated to function as coenzymes in the reaction catalyzed. The purified enzyme was denatured at moderate temperatures and the binding of NADP protected the enzyme against thermal denaturation.