During trophoblast invasion, luminal and glandular endometrial epithelial cells (EEC) have been found to undergo apoptosis through undetermined mechanisms. We postulate that nitric oxide (NO) and progesterone may mediate apoptosis in EEC because they are produced by trophoblasts at concentrations that can cause apoptosis in non-uterine cells. Using a cultured EEC line, RL95-2, we found that sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP), two commonly used NO-releasing agents, caused the death of EEC in a dose-dependent manner and progesterone markedly enhanced NO-induced cytotoxicity. Cells treated with NO/progesterone showed a significant increase in the percentage of condensed nuclei, as detected by DAPI staining, and in caspase-3 activity, indicating that these cells underwent apoptosis. Immunoblot analysis revealed that SNP/NO could activate extracellular signal-regulated kinase (ERK) and, to a lesser extent, p38 mitogen-activated protein kinase (MAPK). While pretreatment with PD98059 (an ERK inhibitor) did not prevent cell death, the addition of SB203580 (a p38 MAPK inhibitor) effectively rescued the cells from NO/progesterone treatment. Moreover, SNP/NO-induced p38 MAPK activation was significantly up-regulated by progesterone. Our results demonstrate that NO and progesterone may synergistically activate p38 MAPK to induce apoptosis in EEC, a process that may facilitate implantation.