The PSD-95 protein family organizes the glutamatergic postsynaptic density and it is involved in the regulation of the excitatory signal at central nervous system synapses. We show here that PSD-95 deficiency by means of antisense oligonucleotides induces significant neuronal cell death within 24 h both in primary hippocampal cultures and in organotypic hippocampal slices. On the other hand, cultured cortical neurons are spared by PSD-95 antisense toxicity until they reach a NR2A detectable protein level (24 days in vitro). The neurotoxic event is characterized by increased alpha CaMKII association to NR2 regulatory subunits of NMDA receptor complex. As a direct consequence of alpha CaMKII association, we found increased GluR1 delivery to cell surface in cultured hippocampal neurons paralleled by AMPA-dependent increase in [Na+]I levels. In addition, both CaMKII specific inhibitor KN-93 and AMPA receptor antagonists CNQX and NBQX rescued neuronal survival to control values. On the other hand, both the NMDA channel blocker MK-801 and Dantrolene, an inhibitor of calcium release from ryanodine-sensitive endoplasmic reticulum stores, failed to have any effect on neuronal survival in PSD-95 deficient neurons. Thus, our data provide clues that PSD-95 reduced expression in neurons is responsible for neuronal vulnerability mediated by direct activation of alpha CaMKII transduction pathway in the postsynaptic compartment.