N-Methyl-D-aspartate causes a rapid increase in intracellular Ca2+ leading to collapse of the mitochondrial membrane potential and eventually cell death in cortical neurons. The aim of this study was to investigate the mechanism responsible for mitochondrial depolarization using laser scanning confocal microscopy of single cultured rate cortical neurons. To monitor mitochondrial membrane potential, neuronal mitochondria were labeled with tetramethylrhodamine methyl ester, a cationic fluorophore that accumulates in polarized mitochondria. In neurons cultured on poly-D-lysine-coated coverslips, N-methyl-D-aspartate caused mitochondrial depolarization in 88% of cells in 30 min. Cyclosporin A, an inhibitor of the mitochondrial permeability transition, delayed depolarization in a dose-dependent manner (0.2-1 microM). In neurons cultured on an astrocyte feeder layer, N-methyl-D-aspartate also caused mitochondrial depolarization. Cyclosporin A again delayed mitochondrial depolarization, although higher concentrations were needed. These data show for the first time that mitochondrial depolarization induced by N-methyl-D-aspartate may be due to the induction of the mitochondrial permeability transition.