The pubertal initiation of spermatogenesis is reliant on androgens, and during this time, 5α-reduced androgens such as dihydrotestosterone (DHT) are the predominant androgens in the testis. Two 5α-reductase (5αR) isoenzymes (5αR1 and 5αR2) have been identified, which catalyze the conversion of testosterone to the more potent androgen DHT. The present study aimed to investigate the developmental pattern of 5αR isoenzymes and their relationship to the production of 5α-reduced androgens in the postnatal rat testis. Both 5αR1 and 5αR2 isoenzyme mRNAs were measured by real-time polymerase chain reaction, isoenzyme activity levels by specific assays, and testicular androgens by radioimmunoassay after high-performance liquid chromatographic separation. Both 5αR1 and 5αR2 mRNAs and activity levels were low in the 10-day-old (prepubertal) testis, peaked between Days 20 and 40 during puberty, and then declined to low levels at 60–160 days of age. The developmental pattern of both 5αR isoenzyme activity levels was mirrored by the testicular production of 5α-reduced metabolites. Although 5αR1 was greater than 5αR2 at all ages, it is likely, given the substrate preferences of the two, that both isoenzymes contribute to the pubertal peak of 5α-reduced androgen biosynthesis. The peak in 5αR isoenzymes and 5α-reduced metabolite production coincided with the first wave of spermatogenesis in the rat, suggesting a role for 5α-reduced metabolites in the initiation of spermatogenesis. This was explored by acute administration of a 5αR inhibitor (L685,273) to immature rats. The L685,273 markedly suppressed testicular 5αR activity during puberty by 75%–86%. However, a marked increase was observed in testicular testosterone levels (in the absence of changes in LH), and no decrease was observed in the absolute levels of 5α-reduced metabolites. Therefore, whether the formation of DHT in the presence of low testosterone levels in the pubertal testis is required for the initiation of spermatogenesis cannot be tested using 5αR inhibitors. We conclude that both 5αR1 and 5αR2 isoenzymes are involved in the peak of 5α-reduced androgen biosynthesis in the testis during the pubertal initiation of spermatogenesis.