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Calcium overload is suggested to play a fundamental role in the process of rod apoptosis in chemical-induced and inherited retinal degenerations. However, this hypothesis has not been tested directly. We developed an in vitromodel utilizing isolated rat retinas to determine the mechanisms underlying Ca2+- and/or Pb2+-induced retinal degeneration. Confocal microscopy, histological, and biochemical studies established that the elevated [Ca2+] and/or [Pb2+] were localized to photoreceptors and produced rod-selective apoptosis. Ca2+and/or Pb2+induced mitochondrial depolarization, swelling, and cytochrome crelease. Subsequently caspase-9 and caspase-3 were sequentially activated. Caspase-7 and caspase-8 were not activated. The effects of Ca2+and Pb2+were additive and blocked completely by the mitochondrial permeability transition pore (PTP) inhibitor cyclosporin A, whereas the calcineurin inhibitor FK506 had no effect. The caspase inhibitors carbobenzoxy-Leu-Glu-His-Asp-CH2F and carbobenzoxy-Asp-Glu-Val-Asp-CH2F, but not carbobenzoxy-Ile-Glu-Thr-Asp-CH2F, differentially blocked post-mitochondrial events. The levels of reduced and oxidized glutathione and pyridine nucleotides in rods were unchanged. Our results demonstrate that rod mitochondria are the target site for Ca2+and Pb2+. Moreover, they suggest that Ca2+and Pb2+bind to the internal metal (Me2+) binding site of the PTP and subsequently open the PTP, which initiates the cytochrome c-caspase cascade of apoptosis in rods.