Some forms of long-term synaptic plasticity require docking of Ca(2+)/calmodulin-dependent protein kinase II α (CaMKIIα) to residues 1290-1309 within the intracellular C-terminal tail of the N-methyl-d-aspartate (NMDA) receptor GluN2B subunit. The phosphorylation of Ser1303 within this region destabilizes CaMKII binding. Interestingly, Ser1303 is a substrate for CaMKII itself, as well as PKC and DAPK1, but these kinases have been reported to have contradictory effects on the activity of GluN2B-containing NMDA receptors. Here, we re-assessed the effect of CaMKII on NMDA receptor desensitization in heterologous cells, as measured by the ratio of steady-state to peak currents induced during 3s agonist applications. CaMKIIα co-expression or infusion of constitutively active CaMKII limits the extent of desensitization and preserves current amplitude with repeated stimulation of recombinant GluN1A/GluN2B when examined using low intracellular chloride (Cl(-)) levels, characteristic of neurons beyond the first postnatal week. In contrast, CaMKIIα enhances the acute rate and extent of desensitization when intracellular Cl(-) concentrations are high. The apparent dependence of CaMKIIα effects on NMDA receptor desensitization on Cl(-) concentrations is consistent with the presence of a Ca(2+)-activated Cl(-) conductance endogenous to HEK 293 cells, which was confirmed by photolysis of caged-Ca(2+). However, Ca(2+)-activated Cl(-) conductances are unaffected by CaMKIIα expression, indicating that CaMKII affects agonist-induced whole cell currents via modulation of the NMDA receptor. In support of this idea, CaMKIIα modulation of GluN2B-NMDA receptors is abrogated by the phospho-null mutation of Ser1303 in GluN2B to alanine and occluded by phospho-mimetic mutation of Ser1303 to aspartate regardless of intracellular Cl(-) concentration. Thus, CaMKII-mediated phosphorylation of GluN2B-containing NMDA receptors reduces desensitization at physiological (low) intracellular Cl(-), perhaps serving as a feed-forward mechanism to sustain NMDA-mediated Ca(2+) entry and continued CaMKII activation during learning and memory.