Activity of the steroid 7±-hydroxylase which converts androstenedione into 7±-hydroxyandrostenedione was detected in testicular microsomal fraction of adult rats, but no significant activity of the enzyme was observed in those of newborn and immature rats. Administration of gonadotropin or testosterone to adult rats reduced the 7±-hydroxylase activity as well as the 17²-hydroxysteroid dehydrogenase in their testis. By experimental bilateral cryptorchidism, the above two enzyme activities were also reduced on the basis of gland, but their specific activities in the microsomal fraction were increased. 7±-Hydroxyandrostenedione and 7±-hydroxytestosterone showed neither androgenic nor anabolic activity in castrated rats. The level of luteinizing hormone in serum elevated by castration was reduced by administration of 7±-hydroxylated C19-steroids. Activities of the adrenal 11²- and 18-hydroxylases, Δ4-5±-hydrogenase and 3±-hydroxysteroid dehydrogenase, that were significantly influenced by castration, recovered to normal level, when testosterone was administered to the castrated animals, but, by administration of 7±-hydroxyandrostenedione to them, no recovery of these activities was observed. From the present experimental results, together with our previous findings, it was concluded that androstenedione was converted in agranular endoplasmic reticula of testicular interstitial cells competitively by 17²-hydroxysteroid dehydrogenase into testosterone and by 7±-hydroxylase into non—androgenic and non—anabolic metabolites. Moreover, 7±-hydroxylated androstenedione inhibited the essential enzyme reactions related to testosterone synthesis from pregnenolone. Testicular 7±-hydroxylation was suggested as a regulatory mechanism of testosterone biosynthesis in rat.(Endocrinology92: 6, 1973)