Astrocyte dysfunction and death accompany cerebral ischemia/reperfusion and possibly compromise neuronal survival. Animal studies indicate that neuronal death, neurologic injury, and oxidative molecular modifications are worse in animals exposed to hyperoxic compared to normoxic ventilation during reperfusion after global cerebral ischemia. It is unknown, however, whether ambient O₂ affects brain cell survival using in vitro ischemia paradigms where mechanisms of injury to specific cell types can be more thoroughly investigated. This study tested the hypothesis that compared with the supraphysiological level of 20% O₂ normally used in cell culture, lower, more physiological O₂ levels protect astrocytes from death following oxygen and glucose deprivation. Primary rat cortical astrocytes were cultured under either 7 or 20% O₂, exposed to O₂, and glucose deprivation for 4 h, and then exposed to normal medium under either 7 or 20% O₂. Cell death and 3‐nitrotyrosine and 8‐hydroxy‐2‐deoxyguanosine immunoreactivities were assessed at different periods of reoxygenation. Astrocytes exposed to low levels of O₂ during reoxygenation undergo less death and exhibit lower levels of protein nitration and nucleic acid oxidation when compared with those under high levels of O₂ during reoxygenation. These results support the hypothesis that the 20% O₂ normally used in cell culture exacerbates astrocyte death and oxidative stress in an in vitro ischemia/reperfusion model compared to levels that more closely approximate those that exist in vivo. © 2008 Wiley‐Liss, Inc.