In this study, it was hypothesized that progesterone (P4) acts as a survival factor primarily by actions of the classic nuclear progesterone receptor (PGR) signaling pathway in rat periovulatory granulosa cells. Granulosa cells were isolated from immature female rats primed with equine chorionic gonadotropin/human chorionic gonadotropin and treated in vitro with PGR antagonists. As little as 10 nM of two different PGR antagonists (Org 31710 and RU 486) increased apoptosis measured as caspase 3/7 activity, which was reversed by cotreatment with the progestin R5020. Concurrently, P4 synthesis was decreased. Inhibition of P4 synthesis by cyanoketone similarly induced apoptosis but required greater inhibition of P4 synthesis than that seen after treatment with PGR antagonists. Therefore, the induction of apoptosis by PGR antagonists cannot be explained by decreased P4 synthesis alone. Low concentrations of R5020 also completely reversed the effects of cyanoketone. Inhibition of P4 synthesis was more effective in inducing apoptosis than treatment with PGR antagonists. However, cotreatment with PGR antagonists protected cells from the additional effects of cyanoketone, indicating partial agonist effects of the antagonists and a dominating role for PGR in P4-mediated regulation of apoptosis. Progesterone receptor membrane component 1 (PGRMC1) was expressed in granulosa cells; however, an anti-PGRMC1 antibody did not induce apoptosis in periovulatory granulosa cells. Neither anti-PGRMC1 nor P4 or cyanoketone affected apoptosis of immature granulosa cells. In conclusion, we show that P4 regulates apoptosis in periovulatory granulosa cells by acting via the classic nuclear receptor.