Ethanol-induced structural changes in membranes have in some studies been attributed to an increase in total membrane cholesterol. Consistent changes in cholesterol content, however, have not been observed in membranes of ethanol consuming animals and alcoholic patients. This study examined the hypotheses that cholesterol was asymmetrically distributed in synaptic plasma membranes (SPM) and that chronic ethanol consumption alters the transbilayer distribution of cholesterol. Dehydroergosterol, a fluorescent cholesterol analogue was used to examine sterol distribution and exchange in chronic ethanol-treated and pair-fed control groups. The cytofacial leaflet was found to have significantly more dehydroergosterol as compared to the exofacial leaflet. This asymmetric distribution was significantly reduced by chronic ethanol consumption as was sterol transport. Total cholesterol content did not differ between the two groups. Chronic ethanol consumption appeared to alter transbilayer sterol distribution as determined by the incorporation and distribution of dehydroergosterol in SPM. The changes in transbilayer sterol distribution are consistent with recent reports on the asymmetric effects of ethanol in vitro ((1988) Biochim. Biophys. Acta 946, 85-94) and in vivo ((1989) J. Neurochem. 52, 1925-1930) on membrane leaflet structure. The results of this study also underscore the importance of examining membrane lipid domains in addition to the total content of different lipids.