DBA/2 mice were made tolerant to and dependent on ethanol by administration of an ethanol-containing liquid diet for 7 days. Fluorescent probe molecules were used to estimate the fluidity and ethanol sensitivity of brain synaptic membranes from these mice. The fluorescence polarization of cis- parinarate , trans- parinarate , and 1,6-diphenyl-1,3,5-hexatriene (probes of the membrane core) and 1-(4- trimethylammoniumphenyl )-6-phenyl-1,3,5-hexatriene (a probe of the membrane surface) was higher in membranes from ethanol tolerant-dependent mice than in membranes from control mice. The decrease in fluorescence polarization produced by in vitro exposure to ethanol was attenuated in membranes from ethanol tolerant-dependent mice when 1,6-diphenyl-1,3,5-hexatriene was used as the probe, but not when 1-(4- trimethylammoniumphenyl )-6-phenyl-1,3,5-hexatriene was used. These results indicate that chronic ingestion of ethanol decreased the fluidity and the ethanol sensitivity of the synaptic membranes. In contrast to the alterations observed with intact membranes, liposomes of lipids extracted from synaptic membranes of control and ethanol tolerant-dependent mice did not differ in their physical properties. Analysis of membrane lipids demonstrated that chronic ethanol treatment selectively decreased the unsaturated acyl groups of phosphatidylserine without altering the acyl composition of other phospholipids or sphingolipids. The amount of each phospholipid was not changed, but membrane cholesterol was decreased by chronic ethanol ingestion. Use of 2-dimensional thin-layer chromatography allowed the quantitation of 10 different gangliosides. The concentrations of these lipids were unchanged in synaptic membranes from ethanol tolerant-dependent mice. Thus, the changes in membrane physical properties produced by chronic ingestion of ethanol may be due, at least in part, to altered acyl composition of phosphatidylserine. The differences observed between intact membranes and extracted lipids suggest, however, that chronic ethanol treatment also produced changes in the lipid arrangement or lipid-protein interactions of the intact membranes.