20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD) from bull testis has been purified to homogeneity and characterized in terms of apparent molecular weight, lack of subunit composition, substrate and cofactor specificity and certain kinetic parameters. The enzyme activity is localized in the 105,000 g supernatant and is stable at 4 degrees C in the presence of glycerol and dithiothreitol. Purification was achieved by ammonium sulfate precipitation followed by affinity chromatography on reactive red 120-agarose and subsequent gel filtration. The apparent molecular weight of the homogeneous enzyme, as determined by gel filtration on Sephacryl S-300 is 34,000. The mobility of the enzyme in sodium dodecyl sulfate (SDS) gel electrophoresis corresponds to a mol. wt of 40,000. These observations indicate that the enzyme is a single-stranded, monomeric polypeptide. The enzyme catalyzes the reduction of the 17-hydroxyprogesterone to 17,20 alpha-dihydroxy-4-pregnene-3-one in the presence of NADPH, the preferred cofactor. Homogeneous 20 alpha-HSD has an SA of 115 mIU/mg, and has been purified 14,000-fold with an overall 68% recovery. It exhibits a pH optimum at 5.6 and appears to be highly specific for 17-hydroxyprogesterone with an apparent Km-value of 7.3 X 10(-5) M. Androstenedione and corticosterone do not serve as substrates under the described experimental conditions. The enzyme does not possess 17 alpha- or 17 beta-HSD activity.