Cerebral ischemia-reperfusion (I/R) injury initiates a cascade of events, generating nitric oxide (NO) and superoxide(O2•-) to form peroxynitrite (ONOO-), a potent oxidant. Arctic ground squirrels (AGS; Urocitellus parryii) show high tolerance to I/R injury. However, the underlying mechanism remains elusive. We hypothesize that tolerance to I/R modeled in an acute hippocampal slice preparation in AGS is modulated by reduced oxidative and nitrative stress. Hippocampal slices (400 μm) from rat and AGS were subjected to oxygen glucose deprivation (OGD) using a novel microperfusion technique. Slices were exposed to NO, O2.- donors with and without OGD; pretreatment with inhibitors of NO, O2.- and ONOO- followed by OGD. Perfusates collected every 15 min were analyzed for LDH release, a marker of cell death. 3-nitrotyrosine (3NT) and 4-hydroxynonenal (4HNE) were measured to assess oxidative and nitrative stress. Results show that NO/O2.- alone is not sufficient to cause ischemic-like cell death, but with OGD enhances cell death more in rat than in AGS. A NOS inhibitor, SOD mimetic and ONOO- inhibitor attenuates OGD injury in rat but has no effect in AGS. Rats also show a higher level of 3NT and 4HNE with OGD than AGS suggesting the greater level of injury in rat is via formation of ONOO-.