BACKGROUND: aCaMKII plays central and essential roles in long-term potentiation (LTP), learning and memory. aCaMKII is activated via binding with Ca²?/CaM in response to elevated Ca²? concentration. Furthermore, prolonged increase in Ca²? concentration leads to the auto-phosphorylation of aCaMKII at T286, maintaining the activation of aCaMKII even after Ca²?/CaM dissociation. Importantly, the active form of aCaMKII is thought to exhibit conformational change. In order to elucidate the relationships between the interaction of aCaMKII with CaM and the conformational change of aCaMKII, we generated molecular probes (YFP-aCaMKII with CFP-CaM and YFP-aCaMKII-CFP) and performed time-lapse imaging of the interaction with CaM and the conformational change, respectively, in living cells using FRET. RESULTS: The interaction of YFP-aCaMKII with CFP-CaM and the conformational change of YFP-aCaMKII-CFP were induced simultaneously in response to increased concentrations of Ca²?. Consistent with previous predictions, high levels of Ca²? signaling maintained the conformational change of YFP-aCaMKII-CFP at the time when CFP-CaM was released from YFP-aCaMKII. These observations indicated the transfer of aCaMKII conformational change from CaM-dependence to CaM-independence. Furthermore, analyses using aCaMKII mutants showed that phosphorylation at T286 and T305/306 played positive and negative roles, respectively, during in vivo interaction with CaM and further suggested that CaM-dependent and CaM-independent conformational changed forms displays similar but distinct structures. CONCLUSIONS: Importantly, these structual differences between CaM-dependent and -independent forms of aCaMKII may exhibit differential functions for aCaMKII, such as interactions with other molecules required for LTP and memory. Our molecular probes could thus be used to identify therapeutic targets for cognitive disorders that are associated with the misregulation of aCaMKII.