The activation of sphingomyelinase and the subsequent generation of ceramide are emerging as important components of signaling pathways leading to apoptosis. The combination of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) induces apoptosis of primary placental trophoblasts in vitro. This apoptosis is inhibited completely by cotreatment with epidermal growth factor (EGF). We therefore examined the role of sphingomyelinase and ceramide in trophoblast apoptosis and how this may be influenced by EGF. Exogenous C16-ceramide (20 microM) and acid sphingomyelinase induced trophoblast apoptosis, an effect abrogated completely by cotreatment with 10 ng/ml EGF. Neutral sphingomyelinase also increased ceramide levels but did not induce apoptosis. Treatment with EGF alone decreased cellular ceramide levels. This decrease could be blocked by cotreatment with the acid ceramidase inhibitor N-oleoylethanolamine (OE). OE alone increased ceramide levels and induced apoptosis that could not be blocked by cotreatment with EGF. In contrast, the alkaline ceramidase inhibitor D-MAPP, although it also increased ceramide levels, did not induce apoptosis nor did it affect TNF-alpha/IFN-alpha-induced cell death. These results implicate sphingolipids as important mediators in trophoblast apoptosis and suggest that the antiapoptotic properties of EGF can in part be explained by its control of ceramide concentrations in trophoblasts.