Perturbed cellular calcium homeostasis has been implicated in both apoptosis and necrosis, but the role of altered mitochondrial calcium handling in the cell death process is unclear. The temporal ordering of changes in cytoplasmic ([Ca2+]C) and intramitochondrial ([Ca2+]M) calcium levels in relation to mitochondrial reactive oxygen species (ROS) accumulation and membrane depolarization (MD) was examined in cultured neural cells exposed to either an apoptotic (staurosporine; STS) or a necrotic (the toxic aldehyde 4-hydroxynonenal; HNE) insult. STS and HNE each induced an early increase of [Ca2+]C followed by delayed increase of [Ca2+]M. Overexpression of Bcl-2 blocked the elevation of [Ca2+]M and the MD in cells exposed to STS but not in cells exposed to HNE. The cytoplasmic calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red prevented both apoptosis and necrosis. STS and HNE each induced mitochondrial ROS accumulation and MD, which followed the increase of [Ca2+]M. Cyclosporin A prevented both apoptosis and necrosis, indicating critical roles for MD in both forms of cell death. Caspase activation occurred only in cells undergoing apoptosis and preceded increased [Ca2+]M. Collectively, these findings suggest that mitochondrial calcium overload is a critical event in both apoptotic and necrotic cell death.