Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a signaling protein required for long-term memory. When activated by Ca2+/CaM, it sustains activity even after the Ca2+ dissipates. In addition to the well-known autophosphorylation-mediated mechanism, interaction with specific binding partners also persistently activates CaMKII. A long-standing model invokes two distinct S and T sites. If an interactor binds at the T-site, then it will preclude autoinhibition and allow substrates to be phosphorylated at the S site. Here, we specifically test this model with X-ray crystallography, molecular dynamics simulations, and biochemistry. Our data are inconsistent with this model. Co-crystal structures of four different activators or substrates show that they all bind to a single continuous site across the kinase domain. We propose a mechanistic model where persistent CaMKII activity is facilitated by high-affinity binding partners that kinetically compete with autoinhibition by the regulatory segment to allow substrate phosphorylation. Display omitted •CaMKII kinase domain binds all interactors through a single continuous binding site•A salt bridge interaction far from active site increases binding affinity•αD helix changes conformation between autoinhibited and active “on” states•High-affinity binders may prolong activation by maintaining the “on” state Özden et al. report high-resolution structures of CaMKII bound to peptides from proteins that are crucial for memory. All interactors dock onto the same single binding site using conserved interactions. A model is proposed for how high-affinity interactors prolong CaMKII activity by competing with autoinhibition.