In previous studies, we have shown that many of the pharmacological effects of ethanol administered in vivo are greater in older mice compared to younger mice. This study determined if there are age-related differences in membrane order when ethanol is administered in vitro. Synaptic plasma membranes, brain microsomal membranes and erythrocyte membranes were isolated from young (3-5 month), middle (11-13 months), and old (22-24 months) C57BL/6NNIA mice. The order parameter of each age group was measured using a 5-nitroxide stearic acid spin label in the presence of 0, 250 and 500 mM ethanol added in vitro. No age-related differences in order parameter were seen in the absence of ethanol. However, membranes from young animals were disordered to the greatest degree by ethanol. The synaptic plasma and erythrocyte membranes from young mice were disordered by both 250 and 500 mM ethanol. Membranes from old mice were disordered significantly by 500 mM ethanol only and the disordering was not as great as was seen in the membranes from the young mice. In the microsomal membrane preparation, 500 mM ethanol significantly disordered the membranes from the young animals, but it had no effect on membranes from old animals. Age differences were also observed generally for cholesterol and total phospholipid which both increased with increasing age for synaptic plasma and brain microsomes. Membrane disorder induced by ethanol differs with age and is associated with cholesterol and phospholipid content of membranes.