To determine whether oxidative stress after cerebral ischemia–reperfusion affects genetic stability in the brain, we studied mutagenesis after forebrain ischemia–reperfusion in Big Blue transgenic mice (male C57BL/6 strain) containing a reporterlacI gene, which allows detection of mutation frequency. The frequency of mutation in this reporter lacI gene increased from 1.5 to 7.7 (per 100,000) in cortical DNA after 30 min of forebrain ischemia and 8 hr of reperfusion and remained elevated at 24 hr reperfusion. Eight DNA lesions that are characteristic of DNA damage mediated by free radicals were detected. Four mutagenic lesions (2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyadenine, 5-hydroxycytosine, and 8-hydroxyguanine) examined by gas chromatography/mass spectrometry and one corresponding 8-hydroxy-2′-deoxyguanosine by a method of HPLC with electrochemical detection increased in cortical DNA two- to fourfold (p < 0.05) during 10–20 min of reperfusion. The damage to γ-actin and DNA polymerase-β genes was detected within 20 min of reperfusion based on the presence of formamidopyrimidine DNA N-glycosylase-sensitive sites. These genes became resistant to the glycosylase within 4–6 hr of reperfusion, suggesting a reduction in DNA damage and presence of DNA repair in nuclear genes. These results suggest that nuclear genes could be targets of free radicals.