The effects of progesterone or 17 alpha-hydroxyprogesterone on corticosterone regulation of beta-endorphin (beta-end) release have been studied in vitro using primary culture of rat anterior pituitaries. Incubation of pituitary cells with ovine corticotropin-releasing factor (CRF) for 2 h resulted in a dose-dependent increase in beta-end release. Maximal stimulation was obtained with 200 ng/ml CRF. Preincubation for 2 h with corticosterone resulted in a dose-dependent inhibition of CRF-induced beta-end release. When the cultures were preincubated for 2 h with 200 ng/ml corticosterone and increasing concentrations (1, 10, 100, 1,000, and 10,000 ng/ml) of progesterone, a significant decrease in the corticosterone feedback action was observed with 100 ng/ml progesterone. Complete inhibition of the action of 200 ng/ml corticosterone was achieved with 10,000 ng/ml progesterone. Moreover, when the cultures were preincubated with increasing concentrations of corticosterone in the presence of 100 ng/ml progesterone, the ED50 of corticosterone increased significantly from 212 +/- 36 to 940 +/- 42 ng/ml (mean +/- SEM; P less than 0.01). Under the same conditions, 17 alpha-hydroxyprogesterone had no effect. These data demonstrate that progesterone antagonizes the corticosterone feedback inhibition of beta-end release by rat anterior pituitary.