The purpose of this study is to determine if calcium/calmodulin-dependent protein kinase-II (CaMKII) plays a role in neuronal cell death and if inhibition of this kinase affords some neuroprotection in the RGC-5 retinal ganglion cell line. The RGC-5 cells were treated with glutamate at various concentrations for increasing increments of time. Cytotoxicity was assayed by measuring the lactate dehydrogenase (LDH) leakage from non-viable cells and TUNEL assays. The involvement of caspase-3, Bcl-2 and caspase-8 in glutamate-induced cytotoxicity was determined by immunoblots and/or real time RT-PCR. In addition, the autocamtide-2-related inhibitory peptide (AIP), a specific inhibitor of CaMKII, was used to determine the involvement of CaMKII in glutamate-induced RGC-5 cell death. Application of increasing concentrations of glutamate to RGC-5 cells caused a dose-dependent increase in the level of cell death after 24 h. There was a glutamate-stimulated increase in the expression of caspase-8 and caspase-3 and a corresponding decrease in Bcl-2. The active fragment of caspase-3 increased in glutamate-treated cells. An early transient increase in the expression of CaMKIIalpha(B) gene and a corresponding CaMKIIalpha nuclear translocation was found in glutamate-treated cells. Treatment with AIP blocked the activation of caspase-3 and protected RGC from glutamate-mediated cell death but did not alter the glutamate-enhanced expression levels of caspase-8 or caspase-3. This report shows the likely involvement of a transcript of the CaMKIIalpha gene in the cytotoxicity response of RGC-5 cells similar to previous reports in the neural retina. AIP is shown to be a neuroprotectant for RGC-5 cells as was reported for the neural retina.