Abstract Here, we show that disruption of N-ethylmaleimide-sensitive fusion protein– (NSF–) GluR2 interaction by infusion into cultured hippocampal neurons of a blocking peptide (pep2m) caused a rapid decrease in the frequency but no change in the amplitude of AMPA receptor–mediated miniature excitatory postsynaptic currents (mEPSCs). N-methyl-D-aspartate (NMDA) receptor–mediated mEPSCs were not changed. Viral expression of pep2m reduced the surface expression of α-amino-3-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptors, whereas NMDA receptor surface expression in the same living cells was unchanged. In permeabilized neurons, the total amount of GluR2 immunoreactivity was unchanged, and a punctate distribution of GluR2 was observed throughout the dendritic tree. These data suggest that the NSF–GluR2 interaction is required for the surface expression of GluR2-containing AMPA receptors and that disruption of the interaction leads to the functional elimination of AMPA receptors at synapses.