delta 4-3-Ketosteroid 5 beta-reductase was purified about 230-fold from 100,000 X g supernatant of rat liver homogenate using 7 alpha-hydroxy-4-cholesten-3-one as substrate throughout. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 37,000 and that determined by gel filtration chromatography on calibrated Sephadex G-100 column was 37,200. The absorption spectrum of the purified enzyme showed only a peak at 276 nm due to aromatic amino acids, precluding the presence of a prosthetic group such as flavine in the molecule. The enzyme is highly labile in a low buffer concentration, but is markedly stabilized in the presence of 20% glycerol in 10 mM phosphate buffer. Higher buffer concentration such as 300 mM potassium phosphate buffer was also effective to prevent deterioration in the absence of glycerol, but the effect was somewhat lower compared to glycerol. The purified enzyme showed the activity toward a variety of substrates including testosterone, cortisol, cortisone, progesterone, 4-androstene-3,17-dione, 7 alpha-hydroxy-4-cholesten-3-one, and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one. The optimal pH for the 5 beta-reduction of 7 alpha-hydroxy-4-cholesten-3-one was 7.4, and the cofactor required for the reaction was NADPH, while NADH revealed no effect. The enzyme activity was inhibited by p-chloromercuribenzoate, but its inhibition was prevented by the presence of a reduced form of glutathione.