For rat pituitary cells, progesterone receptor (PR) protein localizes to gonadotropes and PR messenger RNA is induced by E2 and rapidly but transiently down-regulated by progesterone. Here we quantitatively establish the down-regulatory effect of progesterone on PR protein and evaluate possible mechanisms. Nuclear PR-immunoreactivity (PR-IR) in gonadotropes, identified by dual immunofluorescence, was analyzed by quantitative confocal microscopy. Pituitary cells from female rats were cultured +/- 0.2 nM E2 for 3 days. We confirmed the E2 requirement for PR induction in gonadotropes and determined that the increase in PR-IR required about 24 h. After removal of E2, PR-IR decreases were not found until 24-36 h. Addition of progesterone (40 nM) to E2-treated cells led to a dramatic loss in PR-IR by 9 h (26% of control); by 24 h, PR-IR was barely detectable. Reappearance of nuclear PR-IR required progesterone removal (8-fold increase by 12 h after progesterone removal) and protein synthesis (cycloheximide inhibited the reappearance of PR-IR). Although progesterone decreased PR-IR whether or not E2 was present concurrent with progesterone, the recovery of PR-IR required E2. RU486 completely blocked progesterone-induced PR down-regulation. Because the sustained progesterone-induced loss of PR protein did not correlate with previously reported temporal changes in PR messenger RNA levels, we examined a role for protein degradation. When cells were coincubated with progesterone and the proteasome inhibitor, MG132 (1 microM), the expected decrease in PR protein was abrogated. In summary, progesterone leads to a rapid and extensive reduction in nuclear PR protein in gonadotropes. The progesterone-dependent down-regulation of PR occurs, at least in part, by a proteasome-mediated pathway. Recovery of PR protein requires removal of progesterone, the presence of E2, and protein synthesis. These dynamic changes in nuclear PR levels coincide with the temporal extent of the preovulatory LH surge in rats and could provide a basis for progesterone's biphasic action on LH secretion.