Here, we use a cell surface thrombin cleavage assay to investigate directly the role of NSF in the surface delivery and synaptic accumulation of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors. In cultured hippocampal neurons, the GluR2 subunit (which specifically interacts with NSF) inserts rapidly into the plasma membrane from intracellular compartments and accumulates in synaptic sites. In contrast, surface accumulation of GluR3 (a subunit that does not interact with NSF) or a GluR2 mutant defective in NSF binding (DeltaA849-Q853) occurs initially at extrasynaptic sites and is kinetically slower than wild-type GluR2. Introducing a binding site for NSF into GluR3 (GluR3NSF) generates a subunit that behaves like GluR2 in terms of kinetics and site of surface insertion. These data suggest that the NSF interaction is necessary for rapid incorporation of AMPA receptor subunits into synapses and is sufficient to confer this property on GluR3.