Lactate accumulation in granulosa cell cultures (prepared from estrogen-pretreated immature rat ovaries) increased with human FSH (hFSH) concentration in the culture medium. In 48-h cultures, maximal stimulation (approximately 25%) occurred in the presence of more than or equal to 100 ng hFSH/ml. Human CG (hCG) (3-1000 ng/ml) had no effect. Testosterone and 5 alpha-dihydrotestosterone (10(-8)-10(-6) M) did not affect basal lactate accumulation but they enhanced (dose dependent) the response to hFSH: lactate levels after 48 h of treatment with 10(-7) M testosterone plus 100 ng/ml hFSH were 100% higher than those in untreated control cultures. Lactate was refractory to estradiol and progesterone (10(-8)-10(-6) M) even in the presence of hFSH. Progesterone accumulation showed a qualitatively similar pattern of response to the gonadotropins and sex steroids. As expected, the progesterone response to hFSH (100 ng/ml) plus testosterone (10(-7) M) was progressively suppressed in the presence of 10(-7)-10(-5) M nonsteroidal antiandrogen (SCH16423). Lactate accumulation was also reduced. However, maximal inhibition did not exceed 18% in the presence of SCH16423 at 10(-6) or 10(-5) M as compared with the 80% inhibition of progesterone accumulation observed at 10(-5) M. In the absence of androgenic steroid, the lactate response to hFSH was increased approximately 30% by the high dose of SCH16423. A corresponding synandrogenic action of the drug on FSH-sensitive progesterone accumulation was not observed. These results are evidence that carbohydrate metabolism in differentiating granulosa cells is subject to direct and specific control by FSH and androgenic steroid.