Abstract 1. 1. Properties and kinetics of 3β- Hydroxysteroid dehydrogenase and Δ 5 -3- ketosteroid isomerase from the bovine ovary have been examined. The enzymes were purified from the microsomal fraction utilizing sonication, (NH4)2SO4 precipitation, dialysis, and column chromatography. With DPN+ as cofactor, the purified dehydrogenase oxidizes the 3β- hydroxy position of C19 and C20 steroids of the Δ 4 , Δ 5 , and 5α series. It was inactive with 3α-, 11β-, 17β- and 21- hydroxy positions as well as the 3β- position of cholesterol and did not utilize TPN+ as cofactor. 2. 2. With a Δ 5 -3β- hydroxy compound as substrate there was no significant formation of the Δ 4 -3β- hydroxy compound during incubation without nucleotide cofactor. Thus, the reactions leading to Δ 4 -3- ketones appear to be ordered, with oxidation of Δ 5 -3β- hydroxy compounds to Δ 5 -3- ketones preceding isomerization to Δ 4 -3- ketones . 3. 3. Behaviour during purification indicated that failure to obtain complete solubilization is responsible for lack of success in complete separation of dehydrogenase and isomerase activities. However, non-identity of the sites effecting dehydrogenation and isomerization is indicated by variation of activity ratios during purification, different inactivation rates and different pH curves. 4. 4. The presence of a single dehydrogenase site for C19 and C21 substrates and a single isomerase site for C19 and C20 substrate is indicated by the relative constancy in activity ratios during purification, the kinetics of equimolar mixtures, the competitive activation by alternate substrates, and the similarities of inactivation rates.