To determine if the inhibitory effects of glucocorticoids on GnRH-stimulated secretion of LH observed in male rats in vivo are exerted directly on the pituitary, dispersed pituitary cells from adult male rats were treated with 60 or 600 ng/ml cortisol (F) or corticosterone (B) during one or two 48-h incubations. Control cells received no glucocorticoids. During the second 48 h, some cells from each group were treated with GnRH (2.4 X 10(-11)-6.2 X 10(-8) M). Concentrations of LH and FSH in media and cells were measured by RIA. Treatment with steroids had no effect on basal secretion or maximal GnRH-stimulated secretion of LH, or on maximal secretion of FSH. Treatment with 600 ng/ml B for 96 h increased basal secretion of FSH relative to controls. All treatments with glucocorticoids increased the slopes of the GnRH dose-response curves for both LH and FSH, cell content of LH, total (cells + medium) LH, and total FSH. Incubation with 6 micrograms/ml F or B or 60 ng/ml dexamethasone gave similar results. Decreasing the time period of the second incubation to 6 h results in no significant differences between control cells and cells treated with B or F. These results show that glucocorticoids have different effects in vivo and in vitro, suggesting that inhibitory effects of glucocorticoids on secretion of LH in vivo may not be exerted directly on the pituitary but are exerted elsewhere, perhaps by altered hypothalamic secretion of GnRH. Also, these results show that male and female pituitaries in vitro respond differently to glucocorticoids.