Abstract Guinea pig kidney 17β-hydroxy-C19-steroid dehydrogenase was partially purified by a combination of streptomycin sulfate and ammonium sulfate fractionation, Sephadex filtration, DEAE-cellulose chromatography and a second Sephadex filtration. The specific activity of both TPN- and DPN-linked activities was increased fifty-fold and thirty two-fold for the respective recovered enzyme activities. The purest fraction was increased two hundred thirty-fold in specific activity. The partially purified enzyme sedimented as one symmetrical peak on ultracentrifugation. The s020, w was 3.0S and the D020, w was 7.89×10−7 cm2 sec−1. On disc electrophoresis the TPN- and DPN-linked 17β-OH C19-steroid dehydrogenase activities were revealed in five prominent bands; three weakly stained protein bands showed no enzyme activity. The two cofactor linked activities were not separated throughout the purification steps and had a ratio of 1 to 8 in favor of the TPN-linked activity. The molecular weight was 35,100, 31,600 and 31,200 by ultracentrifugation, Sephadex filtration, and disc electrophoresis respectively. No bound cofactor was detected in the partially purified enzyme fraction. Testosterone produced the highest activity among the steroids tested; 17β-estradiol elicited only slight activity. A crude preparation of the enzyme in 0.25 M sucrose was stable but readily lost activity after purification. Addition of 7mM β-mercaptoethanol and 0.25 M sucrose or 20% glycerol prevented the loss in activity. The enzyme activity was lost on making cytosol 4 M in urea or on dialysis against 8 M urea. The partially purified enzyme was labile to heat but was stable at 4° or −20°. Thiol-blocking agents inhibited the enzyme activity. There were 2.8 moles−SH/mole protein.