Actin filaments are highly concentrated in postsynaptic densities at central excitatory synapses, but their influence on postsynaptic glutamate receptors is unknown. We tested whether actin depolymerization influences NMDA channel activity in whole-cell recording on cultured hippocampal neurons. The ATP- and calcium-dependent rundown of NMDA channels was prevented when actin depolymerization was blocked by phalloidin. Rundown of AMPA/kainate receptors was unaffected by phalloidin. Cytochalasins, which enhance actin-ATP hydrolysis, induced NMDA channel rundown, whereas taxol or colchicine, which stabilize or disrupt microtubule assembly, had no effect. Protease inhibitors also had no effect. Our results suggest that calcium and ATP can influence NMDA channel activity by altering the state of actin polymerization and are consistent with a proposed model in which actin filaments compartmentalize a channel regulatory protein.