Objective: A2A/A1 receptor cross interation and activated A1 receptor activity enhances the inhibition of excitatory toxicity from knock-out A2A receptors in mice ischemic brain. Background Extracellular adenosine level was increased in ischemic brain. A2A receptor inactivation significantly reduces brain injury in the A2A receptor gene knock-out mice. Past research showed no direct inhibition of glutamate release by blocking A2A receptor but only if A1 receptor has been activated first. Design/Methods: MCAO/reperfusion model was created in A2A receptor knock-out (KO) mice and their wild type (WT) for controls. There were three groups: non-treated group, A1 receptor activator treated, and A1 antagonist treated. Changes of neurological function was observed after A1 receptor was stimulated or inactivated. mRNA level of A1 receptor and transporter of glutamate (GLT-1) in ischemic zone were measured(Western-Blot and RT-PCR). All data were analyzed with SPSS 13.0 software. Mann–Whitney U test was used to compare neurological deficit. Results:1. A2A receptor gene knockout can upgrade A1 receptor expression. 2. Ischemic neuroprotection of A2AR deficiency is related to the activation of A1 receptor If untreated, in both A1 antagonist and A1 receptor activator treated group, the neurological deficit scores were lower in KO mice than in WT mice(P Conclusions: The neuroprotection after blocking A2A receptor in ischemic condition is via enhancing the function of the GLT-1 to transport glutamate from extracellular to intracellular space. By up-regulating A1 receptor function, glutamate and its receptor mediated excitatory toxicity have been inhibited. Our research clarified the molecular mechanism of ischemic neuroprotection after A2A receptor has been inhibited. Disclosure: Dr. Gui has nothing to disclose. Dr. Li has nothing to disclose. Dr. Li has nothing to disclose. Dr. Li has nothing to disclose. Dr. Li has nothing to disclose.