Chronic systemic inhibition of mitochondrial respiratory chain complex I by rotenone causes nigrostriatal dopaminergic degeneration in rats, producing an in vivo experimental model of Parkinson's disease. We recently showed that micromolar Ca2+ concentrations strongly stimulate the release of reactive oxygen species in rotenone-treated isolated rat brain mitochondria. In the present work, we show that the natural antioxidant melatonin inhibits Ca2+ plus rotenone-induced oxidative stress in isolated rat brain mitochondria. In addition, the Ca2+ ionophore A23187 strongly potentiates rotenone-induced death of intact cultured pheochromocytoma (PC12) cells, in a mechanism sensitive to melatonin. Moreover, melatonin inhibits the detection of reactive oxygen species release in PC12 cells treated with rotenone plus A23187. Melatonin does not alter free Ca2+ concentrations or the inhibitory effect of rotenone on mitochondrial complex I. We conclude that micromolar Ca2+ concentrations stimulate neuronal cell death induced by mitochondrial complex I inhibition in a mechanism involving oxidative stress, preventable by the antioxidant melatonin.71110-6