Rationale: Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of intracellular signaling in the heart. However, the tools currently available for assessing dynamic changes in CaMKII localization and activation in living myocytes are limited. Objective: We use Camui, a novel FRET-based biosensor in which full-length CaMKII is flanked by CFP and YFP, to measure CaMKII activation state in living rabbit myocytes. Methods and Results: We show that Camui and mutant variants that lack the sites of CaMKII autophosphorylation (T286A) and oxidative regulation (CM280/1VV) serve as useful biosensors for CaMKIIδ activation state. Camui (wild-type or mutant) was expressed in isolated adult cardiac myocytes, and localization and CaMKII activation state were determined using confocal microscopy. Camui, like CaMKIIδ, is concentrated at the z-lines, with low baseline activation state. Camui activation increased directly with pacing frequency, but the maximal effect was blunted with the T286A, consistent with frequency-dependent phosphorylation of CaMKII at T286 mainly at high-frequency and high-amplitude Ca transients. Camui was also activated by 4 neurohormonal agonists. Angiotensin II and endothelin-1 activated Camui, largely through an oxidation-dependent mechanism, whereas isoproterenol- and phenylephrine-mediated mechanisms had a significant autophosphorylation-dependent component. Conclusions: Camui is a novel, nondestructive tool that allows spatiotemporally resolved measurement of CaMKII activation state in physiologically functioning myocytes. This represents a first step in using Camui to elucidate key mechanistic details of CaMKII signaling in live hearts and myocytes.