The present study is to determine the effect of mild hypothermia (MHT) on the release of glutamate and glycine in rats subjected to middle cerebral artery occlusion and reperfusion. The relationship between amino acid efflux and brain infarct volume was compared in different periods during MHT. Reversible middle cerebral artery occlusion was performed in Sprague-Dawley rats using a suture model. The rats were divided into four gruops including (1) MHT during ischemia (MHTi), (2) MHT during reperfusion (MHTr), (3) MHT during ischemia and reperfusion (MHTi+r), and (4) a normothermic group (NT). Extracellular concentrations of glutamate and glycine in the cortex and striatum were monitored using in vivo microdialysis and analyzed using high-performance liquid chromatography. Morphometric for infarct volume were performed using 2, 3.5-triphenyltetrazolium chloride staining. The increase of glutamate and glycine in the ischemic cortex of the MHTi and MHTi+r rats during ischemic and reperfusion periods was significantly less than of the NT rats (p<0.05). However, there was no statistical difference among these groups in the peak of glutamate and glycine release in the striatum. Infarct volume paralleled the release of glutamate and glyeine. The protective effect of MHTi+r reducing ischemia and reperfusion brain injury may be due to the attenuation of both glutamate and glycine release during ischemia and reperfusion.