Calmodulin (CaM) is an essential eukaryotic calcium receptor that regulates many kinases, including CaMKII. Calcium-depleted CaM does not bind to CaMKII under physiological conditions. However, binding of (Ca(2+))(4)-CaM to a basic amphipathic helix in CaMKII releases auto-inhibition of the kinase. The crystal structure of CaM bound to CaMKIIp, a peptide representing the CaM-binding domain (CaMBD) of CaMKII, shows an antiparallel interface: the C-domain of CaM primarily contacts the N-terminal half of the CaMBD. The two domains of calcium-saturated CaM are believed to play distinct roles in releasing auto-inhibition. To investigate the underlying mechanism of activation, calcium-dependent titrations of isolated domains of CaM binding to CaMKIIp were monitored using fluorescence anisotropy. The binding affinity of CaMKIIp for the domains of CaM increased upon saturation with calcium, with the C-domain having a 35-fold greater affinity than the N-domain. Because the interdomain linker of CaM regulates calcium-binding affinity and contribute to conformational change, the role of each CaM domain was explored further by investigating effects of CaMKIIp on site-knockout mutants affecting the calcium-binding sites of a single domain. Investigation of the thermodynamic linkage between saturation of individual calcium-binding sites and CaM-domain binding to CaMKIIp showed that calcium binding to Sites III and IV was sufficient to recapitulate the behavior of (Ca(2+))(4)-CaM. The magnitude of favorable interdomain cooperativity varied depending on which of the four calcium-binding sites were mutated, emphasizing differential regulatory roles for the domains of CaM, despite the high degree of homology among the four EF-hands of CaM.