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3 Beta-Hydroxy-delta-5-steroid dehydrogenase is an important key enzyme of steroid hormone biosynthesis, which is involved in catalyzing the conversion of pregnenolone to progesterone in the biosynthesis of steroid sex hormones. This is the fourth enzyme in the steroid hormone biosynthesis pathway to be identified as an autoantigenic target. Pregnenolone is incubated with several concentrations of whole testis homogenate. The reaction mixture containing the pregnenolone, NAD and iodonitrotetrazolium in 0.15 M Tris-HCL buffer (pH=7.7) and the enzyme extract from testis of rat was incubated for 1.5 h at 36°C. Absorbance at 495 nm was determined by a spectrophotometer. pH optimum and time incubation for this enzyme was 7.99 and 1.5 h respectively. The Kmax was 5.3×10^-6 and Vmax was 3.11×10^-6 nmol/mg/min. Our results were in good agreement with those reported previously.