Cytochrome c is released from mitochondria into the cytosol in cells undergoing apoptosis. The temporal relationship between cytochrome c release and loss of mitochondrial membrane potential was monitored by laser-scanning confocal microscopy in single living pheochromocytoma-6 cells undergoing apoptosis induced by staurosporine. Mitochondrial membrane potential monitored by tetramethylrhodamine methyl ester decreased abruptly in individual cells from 2 to 7 h after treatment with staurosporine. Depolarization was accompanied by cytochrome c release documented by release of transfected green fluorescent protein-tagged cytochrome c in these cells. The results show that mitochondrial depolarization accompanies cytochrome c release in pheochromocytoma-6 cells undergoing apoptosis.