The mechanisms by which neurotransmitter receptors are immobilized at postsynaptic sites in neurons are largely unknown. The activity of NMDA (N-methyl-D-aspartate) receptors is mechanosensitive and dependent on the integrity of actin, suggesting a functionally important interaction between NMDA receptors and the postsynaptic cytoskeleton. alpha-Actinin-2, a member of the spectrin/dystrophin family of actin-binding proteins, is identified here as a brain postsynaptic density protein that colocalizes in dendritic spines with NMDA receptors and the putative NMDA receptor-clustering molecule PSD-95. alpha-Actinin-2 binds by its central rod domain to the cytoplasmic tail of both NR1 and NR2B subunits of the NMDA receptor, and can be immunoprecipitated with NMDA receptors and PSD-95 from rat brain. Intriguingly, NR1-alpha-actinin binding is directly antagonized by Ca2+/calmodulin. Thus alpha-actinin may play a role in both the localization of NMDA receptors and their modulation by Ca2+.