Androgens regulate aromatase activity in the medial preoptic area and other components of the brain circuit that mediates male sexual behavior. The levels of aromatase activity within these brain regions are greater in males than in females. As the activation of copulation requires aromatization of testosterone to estradiol, this quantitative enzymatic difference between sexes could contribute to the greater behavioral response displayed by males. The present study was designed to test the hypothesis that gender differences in brain aromatase activity of adult rats are dependent on the sexual differentiation of the brain that occurs during perinatal exposure to gonadal hormones. Aromatase activity was measured in vitro in microdissected brain samples using a sensitive radiometric assay. We examined the effect of pre- and postnatal treatment with testosterone propionate or diethylstilbestrol on basal levels and androgen responsiveness of aromatase in adults. In addition, we examined what effect prepubertal gonadectomy exerts on enzyme regulation. Our results demonstrate that perinatal treatments with gonadal hormones that are known to differentiate sexual behavior can completely masculinize the capacity for aromatization in the adult female. The process that differentiates aromatase expression appears to depend on androgen exposure and, in part, local estrogen synthesis, as diethylstilbestrol was able to substitute for testosterone propionate. We also observed that prepubertal gonadectomy reduced the levels of aromatase activity measured in adult brain, suggesting that gonadal hormones that are secreted during puberty may enhance the expression of aromatase activity in adulthood. From this study, we conclude that testosterone and/or its estrogenic metabolites act on the developing brain to determine the gender-specific capacity for aromatization and to regulate androgen responsiveness within components of the neural circuitry that mediates male sexual behavior.