Neuronal stretching in culture has been used to model diffuse axonal injury caused by head trauma, and activation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in the pathophysiology of such injury. Here we report the effects of modulating injury severity and the metabotropic glutamate receptor subtype 5 (mGluR5) on NMDAR activity after stretch injury. Following mild stretch, cortical neurons plated upon a confluent layer of astrocytes (NG) exhibited both increased maximal current (I(NMDA)) and reduction in the voltage-dependent Mg2+ block. In contrast, neurons grown without an astrocyte monolayer (PN) only exhibited increased I(NMDA). In NG, surprisingly, pretreatment with either the mGluR5 agonist CHPG or the mGluR5 antagonist MPEP decreased the enhancement of I(NMDA). In contrast, in PN, MPEP similarly limited I(NMDA) changes, but CHPG was without effect. In both culture conditions, MPEP, but not CHPG, limited the stretch-reduced Mg2+ block. Severe stretch had no effect on I(NMDA) or the Mg2+ block in either culture condition, despite a correlation between injury severity and the release of lactose dehydrogenase measured postinjury. Neither CHPG nor MPEP had any direct effects upon the NMDA receptor. We conclude that mGluR5 regulates NMDAR activity during mild stretch injury, but not severe injury, by modulating both the Mg2+ block and I(NMDA).