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 beta-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 microM 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).