Excitatory synaptic activity governs excitotoxicity and modulates the distribution of NMDA receptors (NMDARs) among synaptic and extrasynaptic sites of central neurons. We investigated whether NMDAR localization was functionally linked to excitotoxicity by perturbing F-actin, a cytoskeletal protein that participates in targeting synaptic NMDARs in dendritic spines. Depolymerizing F-actin did not affect NMDA-evoked whole-cell currents. However, the number of dendritic NMDAR clusters and the NMDAR-mediated component of miniature spontaneous EPSCs were reduced, whereas the number of AMPA receptor clusters and AMPA receptor-mediated component of EPSCs was unchanged. This selective perturbation of synaptically activated NMDARs had no effect on neuronal death or the accumulation of (45)Ca(2+) evoked by applying exogenous NMDA or L-glutamate, which reach both synaptic and extrasynaptic receptors. However, it increased survival and decreased (45)Ca(2+) accumulation in neurons exposed to oxygen glucose deprivation, which causes excitotoxicity by glutamate release at synapses. Thus, synaptically and extrasynaptically activated NMDARs are equally capable of excitotoxicity. However, their relative contributions vary with the location of extracellular excitotoxin accumulation, a factor governed by the mechanism of extracellular neurotransmitter accumulation, not the synaptic activation of NMDARs.