N-Methyl- sub(D)-aspartate (NMDA) receptor (NMDAR) activation and downstream signaling are important for neuronal function. Activation of prosurvival Src family kinases and extracellular signal-regulated kinase (ERK) 1/2 is initiated by NMDAR activation, but the cellular organization of these kinases in relation to NMDARs is not entirely clear. We hypothesized that caveolin-1 scaffolds and coordinates protein complexes involved in NMDAR signaling and that this organization is necessary for neuronal preconditioning, whereby NMDAR activation protects neurons from subsequent ischemic cell death. We found that sublethal ischemia (SLI) or preconditioning via NMDA treatment of primary cortical neurons from neonatal rats or mice increases expression of phosphorylated (P) caveolin-1, P-Src, and P-ERK1/2. The NMDAR antagonist, MK801, or the Src inhibitor, PP2, attenuated SLI-induced preconditioning. NMDAR2B distributed to buoyant fractions and heavy fractions, partially colocalized with caveolin-1 and the membrane raft marker, cholera toxin B. Cultures of primary neurons treated with caveolin-1 small interfering RNA or from caveolin-1 mice lacked the NMDA-mediated increase in P-Src and P-ERK, as well as SLI- and NMDA-induced preconditioning. Adenovirally mediated expression of caveolin-1 in neurons from caveolin-1 super(-/-) mice restored NMDA-mediated enhancement of P-Src and P-ERK1/2, redistributed NMDAR2B to buoyant fractions, and enhanced NMDAR2B localization to membrane rafts. We conclude that caveolin-1, perhaps via its ability to scaffold key signaling components, is essential for NMDAR localization to neuronal membrane rafts, NMDAR/Src tyrosine kinase family/ERK signaling, and protection of neurons from ischemic injury and cell death.--Head, B. P., Patel, H. H., Tsutsumi Y. M., Hu, Y., Mejia, T., Mora, R. C., Insel, P. A., Roth, D. M. Drummond, J. C, Patel, P M. Caveolin-1 expression is essential for N-methyl-d-aspartate receptor-mediated Src and ERK 1/2 activation and protection of primary neurons from ischemic cell death.