A mechanism that relates calmodulin (CaM) binding to enzyme activation remains to be established within the context of full-length calmodulin kinase IIalpha (CaM KIIalpha). Previous studies using peptides and/or truncated enzymes have shown that L299 of CaM KIIalpha represents an "anchor" for Ca(2+)/CaM binding and that F293 is required for autoinhibition. We have substituted each of these residues with a W in full-length CaM KIIalpha and measured the W fluorescence to evaluate the location of these side chains in the absence and presence of Ca(2+)/CaM. Fluorescence emission of the L299W mutant indicates that L299 is solvent accessible in the absence of Ca(2+)/CaM but becomes internalized in the presence of Ca(2+)/CaM. On the other hand, examination of F293W indicates that Ca(2+)/CaM binding promotes enzyme activation by transferring F293 from an internal location in the inactive enzyme to a more solvent accessible position in the active enzyme. In addition, F293 interacts with Ca(2+)/CaM as a consequence of autophosphorylation at T286, thus providing a mechanism for CaM trapping. Whereas in the absence of autophosphorylation the exposure of F293 is reversed by dissociation of CaM leading to enzyme autoinhibition, after autophosphorylation of T286, F293 is retained in an exposed position due to dissociation of CaM, consistent with the retention of autonomous activity. Proline mutants were introduced at positions between T286 and F293 to explore the basis of CaM-independent, autonomous activity. The observation that an L290P mutant displayed a high level of activity independent of Ca(2+)/CaM or phosphorylation of T286 indicates that a change in the conformation of the polypeptide main chain at L290 might contribute to the mechanism for generating autophosphorylation-dependent autonomous activity.