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The hormonal regulation of testicular aromatization and the intratesticular site of estradiol synthesis was investigated in adult rats. To determine testicular aromatization, cell-free homogenates of whole testes, isolated interstitial tissue, and seminiferous tubules were incubated with [3H]testosterone. 3H-Labeled estrogens were isolated and final identity was established by recrystallization of the product with authentic 17β-estradiol or estrone. No 3H-labeled estrogens could be identified in testicular incubations from saline-treated rats. In contrast, similar incubations from rats treated for 6 days with hCG or LH had the capacity to aromatize testosterone to estradiol. Testicular homogenates from rats treated with 4.4 IU hCG/day synthesized 4.76 pg estradiol/mg protein, which represents approximately 280 pg estradiol synthesized/testis during a 3-h incubation. In incubations of cell-free homogenates of isolated testicular compartments from hCG-treated rats, aromatase activity was demonstrated only in interstitial tissue and not in seminiferous tubules. LH induction of aromatase activity in testes was time and dose dependent. With two daily injections of 4 μg LH/day, aromatase activity was first detected between 2-4 days, with a marked increase occurring after 6 days of treatment. Administration of increasing doses of LH on a twice daily schedule for 6 days resulted in maximal induction of aromatase at a dose of 4 μg LH/day. In rats treated for 6 days with 2 μg/day of a highly purified preparation of FSH (50 × NIH-FSH-Sl), no testicular aromatization could be demonstrated. The data obtained in this study are consistent with LH or hCG affecting an increase in the synthesis of the aromatase enzyme (s) rather than having an effect on increasing the available substrate, testosterone. This interpretation is based on the following observations: 1) no aromatase activity could be demonstrated in incubations of cellfree testicular homogenates from nontreated rats when testosterone concentration in the incubation medium was increased up to 3 μ.M and 2) rats treated with increasing amounts of LH exhibited a dose-dependent increase in testicular aromatase activity but did not exhibit an increase in endogenous testicular testosterone concentration. We conclude from this study that aromatase activity in the adult rat is regulated by LH and not by FSH.