Testosterone is metabolised to the more potent androgen, dihydrotestosterone, by the 5α-reductase (5αR) enzyme. We previously showed that 5α-reduced androgens are important for maintaining androgen action on rat spermatogenesis when testicular testosterone concentrations are reduced. This study investigated expression and activity of the 5αR isoforms, type 1 (5αR-1) and type 2 (5αR-2), in the rat during hormone manipulation in order to understand the factors that regulate the testicular concentration of 5αR and testicular 5α-reduced androgen biosynthesis. Testicular 5αR-1 and 5αR-2 mRNA and enzyme activity were measured by real-time PCR and specific enzyme assays respectively. Hormone levels were first suppressed using two models of gonadotrophin suppression: testosterone and oestradiol treatment (LH/testosterone deficiency) or GnRH immunisation (LH/testosterone and FSH deficiency). Hormones were then either restored or suppressed for 6 days by a variety of hormonal treatments. 5αR-1 mRNA and enzyme activity increased when testosterone was suppressed, yet restoration of testosterone decreased 5αR-1 mRNA and enzyme activity, suggesting that testosterone negatively regulates 5αR-1. Suppression of FSH decreased 5αR-1 mRNA yet FSH administration increased 5αR-1 mRNA, but no changes in 5αR-1 activity were observed within the 6 day period. In contrast to 5αR-1, testosterone did not affect the testicular concentration of 5αR-2 mRNA or activity, but there was evidence for modulation of 5αR-2 activity by FSH. Measurement of testicular androgens revealed that 5αR-1 was primarily responsible for the production of 5α-reduced metabolites. It is concluded that the 5αR isoforms in rat testis are differentially regulated by testosterone and FSH: testosterone negatively regulated 5αR-1 mRNA and enzyme activity but had no affect on 5αR-2, whereas FSH positively regulated 5αR-1 mRNA and appeared to regulate 5αR-2.