Calmodulin (CaM) functions depend on interactions with CaM‐binding proteins, regulated by Ca2+. Induced structural changes influence the affinity, kinetics, and specificities of the interactions. The dynamics of CaM interactions with neurogranin (Ng) and the CaM‐binding region of Ca2+/calmodulin‐dependent kinase II (CaMKII290−309) have been studied using biophysical methods. These proteins have opposite Ca2+ dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that Ca2+ and CaM interact very rapidly, and with moderate affinity ( KDSPR=3μM). Calmodulin‐CaMKII290−309 interactions were only detected in the presence of Ca2+, exhibiting fast kinetics and nanomolar affinity ( KDSPR=7.1nM). The CaM–Ng interaction had higher affinity under Ca2+‐depleted ( KDSPR=480nM,k1=3.4×105M−1s−1 and k −1 = 1.6 × 10−1s−1) than Ca2+‐saturated conditions ( KDSPR=19μM). The IQ motif of Ng (Ng27−50) had similar affinity for CaM as Ng under Ca2+‐saturated conditions ( KDSPR=14μM), but no interaction was seen under Ca2+‐depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng ( KDMST=890nM) and CaMKII290−309( KDMST=190nM) interactions. Although CaMKII290−309 showed expected interaction characteristics, they may be different for full‐length CaMKII. The data for full‐length Ng, but not Ng27−50, agree with the current model on Ng regulation of Ca2+/CaM signaling.