Testosterone (T) plays an important role in developing brain, dictating sex-specific behavior and physiology. 3α,5α-Reduced neurosteroids also regulate reproductive behavior. The key enzyme in the biosynthesis of these neurosteroids is 5α-reductase (5α-R), expressed as two isozymes, 5α-R1 and 5α-R2. In this study, T and sesame oil (vehicle) were administered during postnatal sexual differentiation of the central nervous system (CNS) and mRNA levels of 5α-R isozymes, were measured using quantitative RT-PCR in prefrontal cortex of male and female rats with different androgenic status at adulthood. Our results indicate that T concentrations during postnatal sexual differentiation of the rat CNS, among other sex-dependent factors, influence brain levels of 5α-R isozymes in adulthood and the pattern of their regulation by androgen hormones.