A rise in intracellular calcium levels ([Ca(2+)](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 [Ca(2+)](i) in retinal ganglion cells (RGCs). Using Ca(2+) imaging techniques we demonstrated that, in the absence of external Mg(2+), the Ca(2+) 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 Ca(2+) channels and AMPA/kainate-Rs contributed a smaller portion of the Ca(2+) response at saturating concentrations of glutamate. Consistent with NMDA-R involvement, extracellular Mg(2+) inhibited RGC glutamate responses, while glycine had a potentiating effect. With Mg(2+) present externally, the effect of AMPA/kainate-R antagonists was enhanced and both NMDA- and AMPA/kainate-R antagonists greatly reduced the glutamate-induced increases of RGC [Ca(2+)](i). This finding indicates that the primary contribution of AMPA/kainate-Rs to RGC glutamatergic Ca(2+) dynamics is through the depolarization-dependent relief of the Mg(2+) block of NMDA-R channels. The effect of glutamate receptor antagonists on glutamatergic Ca(2+) signals from RGCs in adult rat retinal wholemounts yielded results similar to those obtained using immunopanned RGCs. Additional experiments on isolated RGCs revealed that during a 1 h glutamate (10-1000 microm) exposure, 18-28% of RGCs exhibited delayed Ca(2+) deregulation (DCD) and the RGCs that underwent DCD were positive for the death marker annexin V. RGCs with larger glutamate-evoked Ca(2+) 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 ischaemia, and this work establishes a major role for NMDA-R-mediated increases in [Ca(2+)](i) in glutamate-related RGC death.