Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II converts the enzyme to a Ca2+-independent form. The time course for this conversion correlates with the autophosphorylation of a threonine residue located within a thermolytic phosphopeptide common to the alpha and beta/beta' subunits. In the present study, this site was identified in the alpha subunit. After autophosphorylation under conditions that produced near-maximal Ca2+-independent activity, the alpha and beta/beta' subunits were separated by NaDodSO4/PAGE, and the alpha subunit was cleaved with cyanogen bromide. The major phosphopeptide (CB-1), containing phosphothreonine as the only radiolabeled amino acid, was purified by reverse-phase high performance liquid chromatography and subjected to automated gas-phase Edman degradation. The sequence obtained, Xaa-Arg-Gln-Glu-Thr-Val-Asp-Xaa-Leu-Lys-Lys-Phe-Asn-Ala-Arg-Arg-Lys-Leu, represented the NH2-terminal 18 residues (residues 282-299) of a 26-amino acid cyanogen bromide peptide predicted from the deduced primary structure of the alpha subunit and contained a consensus sequence for Ca2+/calmodulin-dependent kinase II phosphorylation that included Thr-286. The sequences obtained for two phosphopeptides derived from secondary chymotryptic digestion of CB-1 confirmed that Thr-286 was the phosphorylated residue.