In order to determine whether androgen acts solely as a substrate for aromatization or whether it also influences on the activity of aromatase enzyme, human granulosa cells were incubated in vitro with or without androgen. Although basal production of 17 beta-estradiol (E2) in cultured granulosa cells obtained from follicles about 26 hr after the initiation of the LH surge was restricted in small quantities, a marked increase in E2 production occurred in the presence of testosterone (T) (10(-6) M) as aromatizable substrate. The non-aromatizable androgen, 5 alpha-dihydrotestosterone (DHT) (10(-7) M, 10(-6) M), slightly enhanced E2 production and it did not inhibit T aromatization in these cells. By contrast, DHT did not increase E2 production in granulosa cells obtained from follicles 2-5 days before the LH surge. The results indicate that androgen can enhance the estrogen biosynthesis of granulosa cells obtained from follicles during the LH surge not only by acting as a substrate for aromatization but also by participating in some process of the estrogen synthesis. This effect of androgen was not clearly seen in granulosa cells obtained before the LH surge, suggesting that the response of granulosa cells to exogenous androgen varies with the stage of the cell differentiation.