Dihydrotestosterone in androgen target tissues is formed under most circumstances by the 5-reduction of testosterone, but an alternate pathway involves the oxidation of androstanediol to dihydrotestosterone. To investigate the mechanism by which androgens virilize the Wolffian ducts in the tammar wallaby, [³H]progesterone was incubated with testes from d 10 and d 19 pouch young, and radioactivity was recovered in testosterone and androstanediol at both ages. Analysis of the intermediates indicates that androstanediol was formed both from testosterone via 5-reduction and 3-keto reduction and directly from 5-reduced progestogens. 5-Reductase activity was high in minces of mesonephros/epididymis from d 6-21 pouch young. When minces of urogenital tract tissues from d 19 pouch young were incubated with [³H]testosterone, [³H]dihydrotestosterone, and [³H]androstanediol, dihydrotestosterone was the principal androgen formed in the mesonephros/epididymis, the urogenital sinus, and the urogenital tubercle whereas androstanediol was the principal androgen formed by the testis. In intact pouch young studied between d 10 and d 34, administration of the 5-reductase inhibitor 4MA blocked virilization of the Wolffian ducts in males, and administration of androstanediol caused virilization of the Wolffian ducts in females. We conclude that dihydrotestosterone, largely formed in the tissue by the oxidation of androstanediol derived from the testes and also from the 5-reduction of testosterone, is responsible for Wolffian duct virilization in this species.