Calmodulin (CaM)-kinase II is inactive in the absence of Ca2+/CaM due to interaction of its autoinhibitory domain with its catalytic domain. Previous studies using synthetic autoinhibitory domain peptides (residues 281-302) identified several residues as important for inhibitory potency and suggested that His282 may interact with the ATP-binding motif of the catalytic domain. To further examine the autoinhibitory domain, site-specific mutants were expressed using the baculovirus/Sf9 cell system. The purified mutants had many biochemical properties identical to wild-type kinase, but mutants H282Q, H282R, R283E, and T286D had 10-20% constitutive Ca(2+)-independent activities, indicating that these residues are involved in the autoinhibitory interaction. The Ca(2+)-independent activities of the H282Q, H282R, and R283E mutants exhibited 10-fold lower Km values for ATP than the wild-type kinase. Wild-type and mutant kinases, except T286A and T286D, generated Ca2+ independence upon autophosphorylation in the presence of Ca2+/CaM, and those mutants having constitutive Ca2+ independence also exhibited enhanced Ca2+/CaM-independent autophosphorylation. This Ca(2+)-independent autophosphorylation resulted in a decrease in total kinase activity, but there was little increase in Ca(2+)-independent activity, consistent with autophosphorylation of predominantly Thr306 rather than Thr286. These results are consistent with an inhibitory interaction of His282 and possibly Arg283 with the ATP-binding motif of the catalytic domain, and they indicate that constitutively active CaM-kinase II cannot autophosphorylate on Thr286 in the absence of bound Ca2+/CaM. Based on these and other biochemical characterizations, we propose a molecular model for the interaction of a bisubstrate autoinhibitory domain with the catalytic domain of CaM-kinase II.