A rise in intracellular calcium levels ([Ca2+]i) is a key trigger for the lethal effects of the excitatory neurotransmitter glutamate in various central neurons, but a consensus has not been reached on the pathways that mediate glutamate-dependent increases of [Ca2+]i in retinal ganglion cells (RGCs). Using Ca2+ imaging techniques we demonstrated that, in the absence of external Mg2+, the Ca2+ signal evoked by glutamate was predominantly mediated by NMDA-type glutamate receptors (NMDA-Rs) in immunopanned RGCs isolated from neonatal or adult rats. Voltage-gated Ca2+ channels and AMPA/kainate-Rs contributed a smaller portion of the Ca2+ response at saturating concentrations of glutamate. Consistent with NMDA-R involvement, extracellular Mg2+ inhibited RGC glutamate responses, while glycine had a potentiating effect. With Mg2+ present externally, the effect of AMPA/kainate-R antagonists was enhanced and both NMDA- or AMPA/kainate-R antagonists greatly reduced the glutamate-induced increases of RGC [Ca2+]i. This finding indicates that the primary contribution of AMPA/kainate-Rs to RGC glutamatergic Ca2+ dynamics is through the depolarization-related relief of the Mg2+ block of NMDA-R channels. The effect of glutamate receptor antagonists on glutamatergic Ca2+ signals from RGCs in adult rat retinal wholemounts yielded similar results to those obtained using immunopanned RGCs. Additional experiments on isolated RGCs revealed that during a 1 hour glutamate (10 - 1000 µM) exposure , 18 - 28% of RGCs exhibited delayed Ca2+ deregulation (DCD) and the RGCs that underwent DCD were positive for the death marker annexin V. RGCs with larger glutamate-evoked Ca2+ signals were more likely to undergo DCD, and NMDA-R blockade significantly reduced the occurrence of DCD. Identifying the mechanisms underlying RGC excitotoxicity aids in our understanding of the pathophysiology of retinal ischemia, and this work establishes a major role for NMDA-R-mediated increases in [Ca2+]i in glutamate-related RGC death.