The effects of ethanol treatment on calcium-binding activity in synaptosomal membrane fraction from rat brains were studied. The synaptosomal membrane fraction from the hippocampus, the cortex and the cerebellum from control, single dose (6 g/kg), dependent intoxicated (prodromal) and dependent withdrawing (ethanol withdrawal syndrome) rats were used. Two different methods were used for determining the calcium activity in these membrane preparations: the calcium chelator fluorescence probe, chlortetracycline (CTC), was used to measure Ca2+ binding sites in the membranes, and radioactive calcium (45Ca) was used to measure the calcium binding to the synaptosomal membranes. Both methods provided similar conclusions; the calcium activity was higher during the dependent intoxicated phase of the ethanol withdrawal period. The synaptosomal membranes from the hippocampus showed more drastic changes in calcium-binding activity than the cortex and the cerebellum. These results suggest that ethanol dependence is associated with changes in calcium-binding activity in certain areas of the rat brain.