The activity of 5α-dihydrotestosterone reduction at 3 positions was mainly observed in the soluble fraction (105, 000×g for 1 hr) from the rat ventral prostate. More than 90% of reduced metabolites were identified as 5α-androstane-3α, 17β-diol when in cubated with various concentrations of the substrate and this may imply that the 3α-reduction of 5α-dihydrotestosterone seems to be a main pathway in this system. Properties of 3α-reduction of 5α-dihydrotestosterone in the soluble fraction from the rat ventral prostate were examined. Optimal pH of the reaction was around 6.4 and required NADPH or NADH. With the latter nucleotide, however, the rate of reduction was lower than that with NADPH. The reaction showed that it was almost the maximum rate with the concentrations of 5α-dihydrotestosterone over 10-5M. The rate of 3α-reduction of 5α-dihydrotestosterone in the ventral prostate was not influenced by castration or testosterone administration. Addition of estradiol-17β to the reaction mixture showed no significant effect on the reaction of 3α-reduction, however testosterone, androst-4-ene-3, 17-dione and cyproterone acetate exhibited an inhibitory effect on the reaction. The soluble fraction from the rat ventral prostate was incubated in the presence of NADPH and testosterone-15C, and the formation of androst-4-ene-3D, 17β-diol-14C was demonstrated.