On exposure to glutamate, cultured rat cerebellar granule cells undergo a delayed Ca2+ deregulation (DCD), which precedes and predicts cell death. We have previously shown that mitochondria control the sensitivity of the neurons to DCD. Mitochondrial depolarization by rotenone/oligomycin before glutamate addition is strongly neuroprotective, and the indication is therefore that mitochondrial Ca2+ loading leads to a delayed loss of bioenergetic function culminating in DCD and cell death. In this report it is shown that superoxide (O2.−) generation in intact cells, monitored by oxidation of hydroethidine to ethidium, was enhanced by glutamate only when mitochondria were polarized. Production of superoxide was higher in the subset of cells undergoing DCD. In the presence of rotenone and oligomycin, addition of glutamate did not result in increased superoxide generation. Menadione-generated superoxide enhances the DCD of cells exposed to glutamate; in contrast, glutamate-induced DCD was potently inhibited by the presence of the cell-permeant antioxidant manganese(III) tetrakis(4-benzoic acid) porphyrin. An inverse correlation is observed between the cytoplasmic free Ca2+ maintained in individual cells in the presence of glutamate and the ability of these cells to restore basal Ca2+ when NMDA receptors are inhibited and mitochondrial Ca2+ is released. It is concluded that mitochondrial Ca2+ accumulation and reactive oxygen species each contribute to DCD, probably related to damage to a process controlling Ca2+ efflux from the cell.