Although the regulation of neurotransmitter receptors during synaptogenesis has been studied extensively at the neuromuscular junction, little is known about the control of excitatory neurotransmitter receptors during synapse formation in central neurons. Using antibodies against extracellular N-terminal (N-GluR1) and intracellular C-terminal (C-GluR1) domains of the AMPA receptor subunit GluR1, combined with surface biotinylation and metabolic labeling studies, we have characterized the redistribution and metabolic stabilization of the AMPA receptor subunit GluR1 during synapse formation in culture. Before synapse formation, GluR1 is distributed widely, both on the surface and within the dendritic cytoplasm of these neurons. The diffuse cell surface pool of receptor appears to be mobile within the membrane and can be induced to cluster by the addition of N-GluR1 to live neurons. As cultures mature and synapses form, there is a redistribution of surface GluR1 into clusters at excitatory synapses where it appears to be immobilized. The change in the distribution of GluR1 is accompanied by an increase in both the half-life of the receptor and the percentage of the total pool of GluR1 that is present on the cell surface. Blockade of postsynaptic AMPA and NMDA receptors had no effect on the redistribution of GluR1. These results begin to characterize the events regulating the distribution of AMPA receptors and demonstrate similarities between synapse formation at the neuromuscular junction and at excitatory synapses in cultured neurons.