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Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the beta2a subunit of voltage-gated Ca2+ channels at Thr498 to facilitate cardiac L-type Ca2+ channels. CaMKII colocalizes with beta2a in cardiomyocytes and also binds to a domain in beta2a that contains Thr498 and exhibits an amino acid sequence similarity to the CaMKII autoinhibitory domain and to a CaMKII binding domain in the NMDA receptor NR2B subunit (Grueter, C. E. et al. (2006) Mol. Cell 23, 641). Here, we explore the selectivity of the actions of CaMKII among Ca2+ channel beta subunit isoforms. CaMKII phosphorylates the beta1b, beta2a, beta3, and beta4 isoforms with similar initial rates and final stoichiometries of 6-12 mol of phosphate per mol of protein. However, activated/autophosphorylated CaMKII binds to beta1b and beta2a with a similar apparent affinity but does not bind to beta3 or beta4. Prephosphorylation of beta1b and beta2a by CaMKII substantially reduces the binding of autophosphorylated CaMKII. Residues surrounding Thr498 in beta2a are highly conserved in beta1b but are different in beta3 and beta4. Site-directed mutagenesis of this domain in beta2a showed that Thr498 phosphorylation promotes dissociation of CaMKII-beta2a complexes in vitro and reduces interactions of CaMKII with beta2a in cells. Mutagenesis of Leu493 to Ala substantially reduces CaMKII binding in vitro and in intact cells but does not interfere with beta2a phosphorylation at Thr498. In combination, these data show that phosphorylation dynamically regulates the interactions of specific isoforms of the Ca2+ channel beta subunits with CaMKII.