Stroke neuroprotection trials suggest that pharmacological manipulations of a single neuroprotective mechanism are generally ineffective and that new approaches, possibly involving simultaneous manipulations of multiple mechanisms, need to be sought. To identify optimal components for such a multipronged approach, we studied NMDA receptor activation-induced cell death in organotypic hippocampal culture preparations as a model of excitotoxicity. Metabotropic group I glutamate receptor (mGluR) activation by their selective agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), resulted in concentration-dependent reduction of nerve cell susceptibility to NMDA-mediated injury (neuroprotective effect). The neuroprotection was mediated primarily by mGluR1, required phospholipase C activation, was inhibited by cholesterol-containing methyl-beta-cyclodextrin treatment, and occluded by antipsychotic quetiapine. It was associated with suppression of NMDA currents and prolongation of GABA(A) receptor-mediated currents in DHPG-treated cultures. cDNA microarray analysis of 1128 brain-relevant genes revealed that mGluR-mediated neuroprotection was associated with simultaneous activation of endocytosis, and inactivation of inflammation, cell adhesion, cell death, and transcription-related genes. Antisense inhibition of Rab5b, a gene coding for a small GTPase associated with endocytosis, significantly reduced the mGluR-mediated neuroprotection. These findings expand our understanding of the role that mGluRs play in regulation of nerve cell susceptibility to injury and should facilitate the design of novel therapeutic strategies for stroke and other neurodegenerative diseases.