Our laboratory found that the N-methyl-D-aspartate receptor (NMDAR) antagonist, MK-801, was able to decrease hyperoxia induced lung damage. To further search for direct evidence of glutamate and its NMDARs participating in hyperoxia-induced lung injury, the amount of glutamate in the bronchoalveolar lavage fluid and the expression of NMDAR 2D in lung tissue were tracked in newborn rats that were exposed to 95% oxygen for 1, 3, and 7 days. The protective effect of MK-801 was then observed at different hyperoxia exposure times. As demonstrated by RT-PCR, NMDAR 2D expression was much higher in hyperoxia exposure on the third and the seventh days than in the air control group. The levels of glutamate in the bronchoalveolar lavage fluid on the first and third days of hyperoxia exposure were significantly higher than in the air control group. MK-801 alleviated lung injury and inflammatory reaction induced by 95% O2 for 3 and 7 days. These results indicate that large amounts of endogenous glutamate from the lungs were released, and its NMDAR were expressed strongly under conditions of high oxygen concentration. We conclude that the endogenous glutamate mediated newborn rat lung damage induced by hyperoxia through NMDARs.