Despite its multifunctional role in cardiac myocyte function, little is known about dynamic changes in activation state of calmodulin (CaM). Thus, the purpose of this study was to develop a tool to measure Ca bound CaM (Ca–CaM) levels in intact cardiac myocytes. For dynamic measurements of Ca–CaM, we generated an adenoviral vector which expresses a cyan and a yellow fluorescent protein linked by a modified version of the Ca–CaM binding domain of avian smooth muscle myosin light chain kinase. Adult rabbit cardiac myocytes were infected with the Ca–CaM sensing probe or simultaneously infected with viruses containing CaM and the Ca–CaM sensing probe for 24–48 h. Myocytes were then field stimulated (1 Hz) and excited at 440 nm with emitted fluorescence measured at 485 and 535 nm. Changes in [Ca–CaM] are expressed as the ratio of 485 nm/535 nm. Small beat-to-beat changes of [Ca–CaM] were detected, but only when CaM was co-expressed with the sensor. However, upon β-adrenergic stimulation with isoproterenol, there was an increase in the amplitude of the signals during each beat (parallel to the shortening, which is an indirect measure of [Ca]_i) and also a rise in the diastolic [Ca–CaM]. Total [CaM] measured by both competitive ELISA and semi-quantitative Western blots was 5–6 μM in isolated adult ventricular myocytes. Our results indicate that there are dynamic changes in free Ca–CaM levels (a phasic component tracking [Ca]_i) as well as system memory that integrates the [Ca]_i signals (a tonic component).