Modifications of the size, shape and number of dendritic spines is thought to be an important component of activity-dependent changes of neuronal circuits, and may play an important role in the plasticity of drug addiction. The present study examined whether homeostatic increases in synaptic N-methyl-d-aspartate (NMDA) receptors in response to chronic ethanol exposure is associated with corresponding morphological changes in dendritic spines. Prolonged exposure of rat hippocampal cultures to either the NMDA receptor antagonist d(–)-2-amino-5-phosphono-pentanoic acid or to ethanol increased punctate staining of F-actin and the postsynaptic density protein-95 (PSD-95). The increase in dendritic F-actin occurred only with clusters that co-localized with PSD-95 clusters, indicating that these actin structures likely represent dendritic spines. The ethanol-induced increases in PSD-95 and F-actin clusters were activity-dependent and reversible. Finally, inhibition of protein palmitoylation prevented ethanol-induced increases in synaptic NMDA receptor clustering and F-actin without altering the basal clustering of either F-actin or PSD-95. These observations support a model in which chronic ethanol exposure induces homeostatic increases of NR2B-containing NMDA receptors and PSD-95 to the postsynaptic density. This in turn may provide a scaffolding platform for the subsequent recruitment of actin signaling cascades that alter actin cycling and promote spine enlargement.