Our previous work showed that ethanol increases the resting intracellular free calcium concentration (CAi) in synaptosomes and releases calcium from an inositol trisphosphate (IP3)-insensitive calcium store of brain microsomes. In this report, we investigated the effects of chronic ethanol treatment and selective breeding for hypnotic sensitivity to ethanol on IP3 and ethanol-stimulated calcium release from brain and liver microsomes. Chronic ethanol treatment did not alter IP3-stimulated calcium release from brain microsomes or ethanol-stimulated calcium release from brain or liver microsomes. Chronic ethanol treatment increased the spontaneous release of calcium from brain but not liver microsomes. In microsomes isolated from cerebellum or cerebral cortex of long-sleep (LS) and short-sleep (SS) mice, ethanol and IP3 released calcium in a concentration dependent manner. The amount of calcium released by ethanol and IP3 was larger in microsomes isolated from cerebellum than microsomes from cerebral cortex. However, the amount of calcium released by ethanol and IP3 did not differ between the two lines in either brain area. These results do not support the idea that the hypnotic effects of ethanol are due to ethanol-induced calcium release from a nonmitochondrial intracellular calcium store in brain tissue. The development of ethanol tolerance and dependence also does not appear to be associated with altered ability of ethanol to release calcium from non-mitochondrial intracellular stores; however, effects of chronic ethanol exposure on spontaneous release of intracellular calcium could alter neuronal function in ethanol dependence.