Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) interacts with several prominent dendritic spine proteins, which have been termed CaMKII-associated proteins. The NR2B subunit of N-methyl-d-aspartate (NMDA)-type glutamate receptor, densin-180, and alpha-actinin bind comparable, approximately stoichiometric amounts of Thr(286)-autophosphorylated CaMKIIalpha, forming a ternary complex (Robison, A. J., Bass, M. A., Jiao, Y., Macmillan, L. B., Carmody, L. C., Bartlett, R. K., and Colbran, R. J. (2005) J. Biol. Chem. 280, 35329-35336), but their impacts on CaMKII function are poorly understood. Here we show that these interactions are differentially regulated and exert distinct effects on CaMKII activity. Nonphosphorylated and Thr(286)-autophosphorylated CaMKII bind to alpha-actinin with similar efficacy, but autophosphorylation at Thr(305/306) or Ca(2+)/calmodulin binding significantly reduce this binding. Moreover, alpha-actinin antagonizes CaMKII activation by Ca(2+)/calmodulin, as assessed by autophosphorylation and phosphorylation of a peptide substrate. CaMKII binding to densin (1247-1542) is partially independent of Thr(286) autophosphorylation and is unaffected by Ca(2+)-independent autophosphorylation or Ca(2+)/calmodulin. In addition, the CaMKII binding domain of densin-180 has little effect on CaMKII activity. In contrast, the interaction of CaMKIIalpha with NR2B requires either Thr(286) autophosphorylation or the binding of both Ca(2+)/calmodulin and adenine nucleotides. NR2B inhibits both the Ca(2+)/calmodulin-dependent and autonomous activities of CaMKII by a mechanism that is competitive with autocamtide-2 substrate, non-competitive with syntide-2 substrate, and uncompetitive with respect to ATP. In combination, these data suggest that dynamically regulated interactions with CaMKII-associated proteins could play pleiotropic roles in finetuning CaMKII signaling in defined subcellular compartments.