Mice were treated with ethanol for eight or nine days, using a liquid diet regimen known to produce physical dependence. In previous experiments, synaptosomal plasma membranes and erythrocyte ghosts from such ethanol-treated animals were found to be resistant to the fluidizing effects of ethanol in vitro, as measured by electron paramagnetic resonance. In the present experiments, corresponding membranes were analysed for phospholipid and cholesterol. The ratio of cholesterol to phospholipid was found to be significantly increased in both types of membrane after chronic ethanol treatment. The changed ratio was produced by an increase in cholesterol. There was little or no change in phospholipid content of the membranes. Increased cholesterol may explain the previously observed alteration of physical properties of the membranes.