The role of androgen in aromatase induction/activation by follicle-stimulating hormone (FSH) was studied in cultured granulosa cells from estrogen-pretreated, immature rat ovaries. Aromatase activity was measured in washed cell monolayers after a 48-h culture in medium containing hFSH and/or various sex steroids or their analogues. Culture with hFSH (100 ng/ml) plus 10(-7) M testosterone (T) stimulated aromatase activity to a level similar to that of granulosa cells from preovulatory follicles in the cyclic adult on the morning of proestrus. But if T was omitted, or replaced by estrogen (DES) or progesterone (P), the response to hFSH was at least 90% lower. The abilities of T, androstenedione, five nonaromatizable 5 alpha-reduced androgens, an aromatase reaction intermediate (19-hydroxyandrostenedione), and a pharmacological competitive aromatase inhibitor (delta 1-testoloalactone) to stimulate the aromatase response to hFSH were proportionate to their stimulatory effects on P production during the culture. By both criteria T was the most potent androgen while 19-hydroxyandrostenedione and delta 1-testololactone were completely inactive. The stimulatory effect of 10(-7) M T on the aromatase response to FSH was inhibited by the nonsteroidal antiandrogen SCH 16423 (ID50 = 3.6 x 10(-6) M). These results indicate that granulosa cell aromatase induction/activation by hFSH is an androgen receptor-regulated process in vitro.