Gonadectomy results in a striking sex difference in plasma LH levels in adult rats; by 2 days postgonadectomy, serum LH is 5- to 10-fold higher in males than in females. Despite 25 years of work, the site of this sex difference has yet to be determined. Since gonadectomy elevates GnRH pulse frequency, we asked whether a sex difference in pituitary LH secretory response to GnRH pulse frequency underlies the sex difference in serum LH postgonadectomy. Pituitaries from intact and 2-day postgonadectomized male and female rats were perifused for 8 h, receiving either no GnRH (basal) or GnRH pulses (50 ng/ml) at 1 pulse/1.5 h, 1/h or 2/h. Five-minute perifusate fractions were analyzed for LH, FSH, and PRL by RIA. At 1 GnRH pulse/1.5 h, but not other frequencies, pituitaries from gonadectomized males released 1.4-2.8 times more LH per pulse than female pituitaries. Such in vitro sex differences in LH release were not due to higher in vitro prolactin release by female than male pituitaries as suppression of pituitary prolactin secretion by perifusion with the dopamine agonist bromocriptine failed to erase sex differences. Since GnRH pulse frequency in vivo exceeds 1/h following castration, it is unlikely that weak sex differences at frequencies slower than 1/h are relevant to differences in serum LH. In conjunction with prior pituitary studies in this laboratory, these data conclusively demonstrate that the pituitary is not the site of acute sex differences in serum LH levels following gonadectomy. Previous in vitro work indicates that higher GnRH amplitudes elicit higher gonadotropin release, suggesting that differences in the relative amounts of GnRH released by male and female hypothalami may underlie the gender differences observed in serum LH levels immediately postgonadectomy.