This study examined the effects of maintaining cells in different media and the role of serum in glutamate and NMDA-induced neurotoxicity in rat cerebellar granule cells. Glutamate stimulated a concentration-dependent cell death with similar potency in cerebellar granule cells grown in BME and Neurobasal media without serum. However, the maximal cell death to glutamate and N-methyl-D-aspartate (NMDA) varied in the different media compositions. In the presence of serum, glutamate and NMDA-induced excitotoxicity was abolished, suggesting a factor(s) in serum which influences glutamate-receptor mediated death. The protective effect of serum could be overcome by chronic stimulation with high doses of glutamate. The glutamate-stimulated increase in intracellular calcium load was attenuated in the presence of serum, resulting from an elevated basal calcium level, suggesting an association between raised basal calcium and neuroprotection.