Recent studies have demonstrated that adenosine A(2A) receptor (A(2A)R) inactivation protects against brain injury caused by cerebral ischemia in various animal models. However, the underlying mechanisms remain to be fully elucidated. We examined the effect A(2A)R genetic inactivation on extracellular glutamate in the striatum and its relationship to the neuroprotection afforded by A(2A)R inactivation following transient middle cerebral artery occlusion (MCAo) in mice. Extracellular glutamate in the striatum was collected by in vivo microdialysis during cerebral ischemia and after reperfusion, and then determined with high-performance liquid chromatography. We found that the glutamate level was indistinguishable between A(2A)R knock-out (A(2A)R-KO) mice and their wild type (A(2A)R-WT) littermates before MCAo. After MCAo a remarkable increase in the glutamate level was observed in the A(2A)R-WT mice, but the increase in glutamate level was significantly attenuated in the A(2A)R-KO mice. The cerebral reperfusion induced a second wave of increase of the glutamate level in the A(2A)R-WT mice, and again this increase was largely attenuated in the A(2A)R-KO mice. Correlating with attenuated glutamate level, the neurological deficits and the cerebral infarct volume were also significantly reduced in the A(2A)R-KO mice compared with their WT littermates. These results demonstrate that the genetic inactivation of A(2A)R inhibits the glutamate outflow and ameliorates the brain injury in both ischemic and reperfusion phases in the transient focal cerebral ischemia model. It suggests that the protection of A(2A)R inactivation against ischemic brain injury is associated with the suppression of glutamate-dependent toxicity.