BACKGROUND The central nervous system is particularly sensitive to the cytotoxic effect of ethanol. In vivo and in vitro studies indicate that ethanol decreases cell proliferation in a number of cells types, including neurons and glial cells in the central nervous system. The cellular mechanisms involved in ethanol-induced cell toxicity, however, are unclear. In this study, we examined the effect of ethanol on tumor necrosis factor-alpha (TNFalpha)-induced cell death in a homogeneous population of cultured rat astrocytes. METHODS Flow cytometric and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) dye reduction analyses were performed on cultured rat astrocytes to determine the effect of alcohol on TNFalpha-induced cell death. RESULTS Flow cytometric analysis revealed that, in quiescent astrocytes, high concentrations of ethanol were required to increase DNA fragmentation and decrease cell viability. Preexposure of astrocytes to low concentrations of ethanol (10 to 50 mM), however, increased the sensitivity of astrocytes to TNFalpha with low TNFalpha concentrations (25 to 50 ng/ml) resulting in increased DNA fragmentation. Furthermore, MTT dye reduction analysis revealed that exposure of astrocytes to 5 mM ethanol was sufficient to increase the susceptibility of astrocytes to the cytotoxic effect of ethanol. In a number of cell types, TNFalpha receptor binding results in the activation of specific signal transduction cascades, including the hydrolysis of sphingomyelin to ceramide. We show that preexposure of astrocytes to a low concentration of ethanol increased the sensitivity of astrocytes to sphingomyelinase, and C2-ceramide resulting in increased DNA fragmentation and decreased cell viability. More importantly, astrocytes prepared from rats exposed to ethanol prenatally showed increased susceptibility to TNFalpha-induced cell death. CONCLUSIONS These studies suggest that ethanol increases the susceptibility of astrocytes to TNFalpha-induced cell death by shifting the balance of sphingolipid metabolism in favor of a pathway that increases the susceptibility of astrocytes to the cytotoxic effect of TNFalpha.