The effects of ethanol in vitro on Ca(++)-dependent binding of [125I] calmodulin to brain synaptic plasma membranes (SPM) from control and chronically ethanol-treated rats were studied. In SPM from control animals, ethanol at 50 to 200 mM inhibited [125I] calmodulin binding; the inhibition was correlated with a decreased membrane affinity for [125I]calmodulin as shown by Scatchard analysis, and an increased dissociation of [125I]calmodulin-membrane complexes as shown by kinetic analysis. Arrhenius analysis indicates that [125I]calmodulin binding was influenced by lipid transition of the membrane, and that ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were found to be resistant to the inhibitory effect of ethanol on binding. The resistance to ethanol inhibition was correlated with a higher membrane affinity for [125I]calmodulin and a higher transition temperature, as compared with control SPM. Because a variety of membrane-bound processes are regulated by calmodulin or calmodulin-dependent processes, the inhibitory effect of ethanol on membrane binding of calmodulin could lead to a cascade of consequences in synaptic function. Moreover, the resistance of the membranes to ethanol inhibition after chronic ethanol treatment implies that membrane binding of calmodulin is part of the mechanism underlying alcohol tolerance and dependence.