The present study was undertaken to investigate the effects of ceramide on progesterone production and apoptosis in rat luteal cells in vitro. Luteal cells were prepared from the ovaries of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) primed female Wistar rats and incubated with cell permeable C2-ceramide. The effects of ceramide on progesterone production and apoptosis in luteal cells were assessed by radio immunoassay (RIA) and flow cytometry analysis. In addition, changes in nitric oxide synthase (NOS) activity and nitric oxide (NO) production by luteal cells treated with C2-ceramide were also evaluated. Ceramide was found to reduce hCG-stimulated progesterone production in a dose-dependent manner, whereas it had little effect on basal progesterone content. Spontaneous apoptosis in luteal cells was observed after a 12-hour incubation in vitro and 5 mumol/L of ceramide significantly increased the apoptotic rate (P< 0.05). Enhanced apoptotic peak was seen in the histogram by flow cytometry. Moreover, 50 mumol/L of C2-ceramide significantly increased NOS activity (P< 0.01) and NO production (P< 0.001). It is suggested that ceramide may serve as an important signaling molecule mediating certain ovarian processes, such as luteal regression.