Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the β_2a subunit of voltage-gated Ca²⁺ channels at Thr498 to facilitate cardiac L-type Ca²⁺ channels. CaMKII colocalizes with β_2a in cardiomyocytes and also binds to a domain in β_2a 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 β subunit isoforms. CaMKII phosphorylates the β_1b, β_2a, β₃, and β₄ isoforms with similar initial rates and final stoichiometries of 6−12 mol of phosphate per mol of protein. However, activated/autophosphorylated CaMKII binds to β_1b and β_2a with a similar apparent affinity but does not bind to β₃ or β₄. Prephosphorylation of β_1b and β_2a by CaMKII substantially reduces the binding of autophosphorylated CaMKII. Residues surrounding Thr498 in β_2a are highly conserved in β_1b but are different in β₃ and β₄. Site-directed mutagenesis of this domain in β_2a showed that Thr498 phosphorylation promotes dissociation of CaMKII-β_2a complexes in vitro and reduces interactions of CaMKII with β_2a in cells. Mutagenesis of Leu493 to Ala substantially reduces CaMKII binding in vitro and in intact cells but does not interfere with β_2a phosphorylation at Thr498. In combination, these data show that phosphorylation dynamically regulates the interactions of specific isoforms of the Ca²⁺ channel β subunits with CaMKII.