The synthetic pathway by which 5α-androstane-3α,17β-diol (5α-adiol) is formed in the testes of tammar wallaby pouch young was investigated by incubating testes from d 20–40 males with various radioactive precursors and analyzing the metabolites by thin-layer chromatography and HPLC. [³H]Progesterone was converted to 17-hydroxyprogesterone, which was converted to 5α-adiol by two pathways: One involves the formation of testosterone and dihydrotestosterone as intermediates, and the other involves formation of 5α-pregnane-3α,17α-diol-20-one (5α-pdiol) and androsterone as intermediates. Formation of 5α-adiol from both [³H]testosterone and [³H]progesterone was blocked by the 5α-reductase inhibitor 4MA. The addition of nonradioactive 5α-pdiol blocked the conversion of [³H]progesterone to 5α-adiol, and [³H]5α-pdiol was efficiently converted to androsterone and 5α-adiol. We conclude that expression of steroid 5α-reductase in the developing wallaby testes allows formation of 5α-reduced androgens by a pathway that does not involve testosterone as an intermediate.