Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase) induces a more than 1000-fold increase in calmodulin (CaM)-binding affinity by dramatically decreasing the off-rate for CaM. In this report, we investigate the molecular mechanism for this phenomenon by comparing the rate of dissociation of a novel fluorescently labeled CaM from two synthetic peptides and from the phosphorylated and nonphosphorylated forms of a recombinant preparation of CaM-kinase. Dissociation of a complex of CaM and CKII(296-312), a peptide representing close to the minimum CaM-binding domain of the alpha subunit of CaM-kinase, exhibited a fast off-rate of 5.0 s-1. This was similar to the off-rate of 1.1 s-1 for the dissociation of CaM from the nonphosphorylated form of CaM-kinase. In contrast, dissociation of CaM from either autophosphorylated CaM-kinase or peptide CKII(290-314) was extremely slow with apparent off-rates of about 3-9 x 10(-5) s-1. Along with information from the crystal structure of Ca2+/CaM bound to CKII(290-314) (Meador, W. E., Means, A. R., and Quiocho, F. A. (1993) Science 262, 1718-1721), our results suggest a model in which CaM-dependent autophosphorylation of CaM-kinase induces a conformational change in the region of the CaM-binding domain which allows the formation of additional stabilizing interactions with CaM. We predict that this involves amino acids 293-298 in CaM-kinase. The possible consequences of these observations on the reversibility of CaM trapping in native CaM-kinase are discussed.