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Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca 2+ /CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca 2+ /CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca 2+ /CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca 2+ /CaM and not to CaMKII. This binding would disrupt the ability of Ca 2+ /CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca 2+ /CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca 2+ /CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca 2+ /CaM-dependent, non-CaMKII activities should be considered in KN-93-based mechanism-of-action studies and drug discovery efforts., (Copyright © 2019 Elsevier Ltd. All rights reserved.)