The effects of glutamate on intracellular free Ca2+ [Ca2+]i, and neurotoxicity were compared in cerebellar granule neurons in vitro. [Ca2+]i, was measured with fura-2 and digital fluorescence imaging microscopy; neurotoxicity was monitored using a vital dye and colorimetric analysis. Glutamate produced dosedependent increases in [Ca2+]XSi transient for glutamate concentrations in a range of : 0.01–0.5 μM and sustained for higher levels of glutamate. The ED50 for the [Ca2+]i response to glutamate was. 6 μM. The LQ50 for glutamate-induced neurotoxicity was similar, i.e., 10 μM The effect of glutamate on [Ca2+]i was greatly dmiinished when external Ca2+ was removed and blocked by Mg2++ N-methyl-D-aspartate (NMDA)-type receptor antagonists. The latter conditions as well as preloading granule neurons with the intracellular Ca2+ chelator quin2 largely prevented glutamate cytotoxicity. The neurotoxic effect of glutamate required incubations with the stimulus for 10–20 min at 25°C. Withdrawal of glutamate after this period was accompanied by a prolonged alteration in [Ca2+]i. Pretreatment cells with the ganglioside GM1 reduced this late increase in [Ca2+]i, as well as the neurotoxic effects of glutamate. This indicates that glutamate-induced neurotoxicity results from a composite of diverse temporal alterations in Ca2+ homeostasis and that blunting any of these components reduces excitotoxicity.