Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol-induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol-induced increase in intracellular calcium [Ca2+]i causes the death of CGN. Alcohol induced a dose-dependent (200–800 mg/dl) neuronal death within 24 hours. Ratiometric Ca2+ imaging with Fura-2 revealed that alcohol causes a rapid (one-two minutes), dose-dependent increase in [Ca2+]i, which persisted for the duration of the experiment (5 or 7 minutes). The alcohol-induced increase in [Ca2+]i was observed in Ca2+-free media, suggesting intracellular Ca2+ release. Pre-treatment of CGN cultures with an inhibitor (2-APB) of the inositol-triphosphate receptor (IP3R), which regulates Ca2+ release from the endoplasmic reticulum (ER), blocked both the alcohol-induced rise in [Ca2+]i and the neuronal death caused by alcohol. Similarly, pre-treatment with BAPTA/AM, a Ca2+-chelator, also inhibited the alcohol-induced surge in [Ca2+]i and prevented neuronal death. In conclusion, alcohol disrupts [Ca2+]i homeostasis in CGN by releasing Ca2+ from intracellular stores, resulting in a sustained increase in [Ca2+]i. This sustained increase in [Ca2+]i may be a key determinant in the mechanism underlying alcohol-induced neuronal death.