Oxidative stress and DNA oxidation play important roles in the induction of ischemic neuronal cell death. However, the subcellular source of oxidized DNA detected by 8-hydroxy-2'-deoxyguanosine (8-OHdG) after ischemia has not been clarified although it is known to increase in the brain after ischemia. One-hour transient ischemia of the middle cerebral artery was induced in mice utilizing an intraluminal filament. The occurrence of superoxide anion as an ethidium (Et) signal, 8-OHdG, cytochrome c release and neuronal cell death were examined using immunohistological and biochemical techniques in sham-operated control (0h) and 1, 3, 6, 24, or 96h after reperfusion. Et signals were prominent in the cortical neurons of ipsilateral hemisphere 3h after reperfusion. Strong 8-OHdG immunoreactivity was observed 3-6h after reperfusion. Immunoassays after cell fractionation revealed a significant increase of 8-OHdG in mitochondria 6h after reperfusion. Immunohistochemistry revealed that the 8-OHdG immunoreactivity colocalized with a neuronal marker, microfilament 200 and a mitochondrial marker, cytochrome oxidase subunit I. Cytochrome c rose in cytoplasm at 6h and TUNEL-positive neurons noted 6-24h after ischemia. The present results suggest the possibility that the mitochondrial damage including mitochondrial DNA oxidation might be responsible for the induction of ischemic neuronal cell death.