Calcium-calmodulin-dependent protein kinase II (CaMKII) is thought to increase synaptic strength by phosphorylating postsynaptic density (PSD) ion channels and signaling proteins. It is shown that N-methyl-D-aspartate (NMDA) receptor stimulation reversibly translocates green fluorescent protein-tagged CaMKII from an F-actin-bound to a PSD-bound state. The translocation time was controlled by the ratio of expressed [small beta, Greek]-CaMKII to [small alpha, Greek]-CaMKII isoforms. Although F-actin dissociation into the cytosol required autophosphorylation of or calcium-calmodulin binding to [small beta, Greek]-CaMKII, PSD translocation required binding of calcium-calmodulin to either the [small alpha, Greek]- or [small beta, Greek]-CaMKII subunits. Autophosphorylation of CaMKII indirectly prolongs its PSD localization by increasing the calmodulin-binding affinity.