Apoptosis of retinal ganglion cells (RGCs) impairs vision in glaucoma patients. RGCs are also degenerated in multiple sclerosis (MS), resulting in loss of visual perception in MS patients. We examined the involvement of calpain and caspase cascades in apoptosis of the rat retinal ganglion cell line RGC-5 following 24 h of exposure to 250 nM ionomycin (IMN) or 300 units/ml interferon-gamma (IFN-gamma) and then evaluated functional neuroprotection with 2 microM calpeptin (CP, a calpain-specific inhibitor). Morphological and biochemical features of apoptosis were detected in RGC-5 cells following exposure to IMN or IFN-gamma. Fura-2 assay determined significant increases in intracellular free [Ca2+] following exposure to IMN or IFN-gamma. Pretreatment with CP for 1 h prevented Ca2+ influx, proteolytic activities, and apoptosis in RGC-5 cells. Western blot analyses showed an increase in activities of calpain and caspase-12, upregulation of Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and increase in caspase-9 and caspase-3 activities during apoptosis. Increased caspase-3 activity was also confirmed by a colorimetric assay. Activation of caspase-8 and cleavage of Bid to tBid in RGC-5 cells following exposure to IFN-gamma indicated co-operation between extrinsic and intrinsic pathways of apoptosis. Patch-clamp recordings showed that pretreatment with CP attenuated apoptosis and maintained normal whole-cell membrane potential, indicating functional neuroprotection. Taken together, our results demonstrated that Ca2+ overload could be responsible for activation of calpain and caspase cascades leading to apoptotic death of RGC-5 cells and CP provided functional neuroprotection.