Cardiac myocyte intracellular calcium varies beat-to-beat and calmodulin (CaM) transduces Ca2+ signals to regulate many cellular processes (e.g. via CaM targets such as CaM-dependent kinase and calcineurin). However, little is known about the dynamics of how CaM targets process the Ca2+ signals to generate appropriate biological responses in the heart. We hypothesized that the different affinities of CaM targets for the Ca2+-bound CaM (Ca2+-CaM) shape their actions through dynamic and tonic interactions in response to the repetitive Ca2+ signals in myocytes. To test our hypothesis, we used two fluorescence resonance energy transfer-based biosensors, BsCaM-45 (Kd = approximately 45 nm) and BsCaM-2 (Kd = approximately 2 nm), to monitor the real time Ca2+-CaM dynamics at low and high affinity CaM targets in paced adult ventricular myocytes. Compared with BsCaM-2, BsCaM-45 tracks the beat-to-beat Ca2+-CaM alterations more closely following the Ca2+ oscillations at each myocyte contraction. When pacing frequency is raised from 0.1 to 1.0 Hz, the higher affinity BsCaM-2 demonstrates significant elevation of diastolic Ca2+-CaM binding compared with the lower affinity BsCaM-45. Biochemically detailed computational models of Ca2+-CaM biosensors in beating cardiac myocytes revealed that the different Ca2+-CaM binding affinities of BsCaM-2 and BsCaM-45 are sufficient to predict their differing kinetics and diastolic integration. Thus, data from both experiments and computational modeling suggest that CaM targets with low versus high Ca2+-CaM affinities (like CaM-dependent kinase versus calcineurin) respond differentially to the same Ca2+ signal (phasic versus integrating), presumably tuned appropriately for their respective and distinct Ca2+ signaling pathways.