At least two 20α-faydroxysteroid dehydrogenases occur in the dialyzed high-speed supernatant fraction of mouse liver. These enzymes have been separated from each other with good yields of each and some of their kinetic properties have been studied. With corticosterone, 11-dehydrocorticosterone, and 11β-hydroxyprogesterone as substrates, it has been shown that the specificity of each enzyme is determined, in part, by the nature of the substituent at C-21, hence the enzymes have been designated 21-desoxy- or 21-hydroxy-20α-hydroxysteroid 20α-dehydrogenase. An enzymatic method is described for the preparation of 11β,20α-dihydroxy-4-pregnen-3-one.