Release of cytochrome c from mitochondria to the cytosol is a critical step in apoptotic cell death after focal cerebral ischemia. The relationship among cytochrome c release, selective vulnerability, and delayed death of hippocampal CA1 neurons after transient global ischemia was examined. Global ischemia was induced by 10 min of bilateral common carotid artery occlusion and hypotension in rats. Cytosolic expression of cytochrome c was evaluated by immunohistochemistry and Western blotting. Apoptosis after global ischemia was also characterized by terminal deoxynucleotidyl transferase-mediated uridine 5-triphosphate-biotin nick end-labeling (TUNEL) staining and DNA gel electrophoresis. Immunohistochemistry showed cytosolic cytochrome c-positive cells exclusively in the CA1 subregion of the hippocampus as early as 2 hr after ischemia. Double fluorescent immunostaining confirmed that CA1 neurons and a small number of astrocytes expressed cytochrome c. Western blot analysis revealed a band (15 kDa) of cytochrome c in the cytosolic fraction and a corresponding decrease in the mitochondrial fraction. A significant number of TUNEL-positive cells appeared only in the CA1 pyramidal cell layer of the hippocampus, and DNA gel electrophoresis showed a significant amount of DNA fragmentation 3–5 d after ischemia. Our data provide the first evidence that cytochrome c was released to the cytosol from mitochondria in CA1 neurons after global ischemia and that the release preceded DNA fragmentation. These findings suggest cytochrome c involvement in the delayed death of hippocampal CA1 neurons in rats after transient global ischemia.