BACKGROUND: Application of fluorescence imaging of cardiac electrical activity is limited by motion artifacts and/or side effects of currently available pharmacologic excitation–contraction uncoupling agents. OBJECTIVES: The purpose of this study was to test whether blebbistatin, a recently discovered inhibitor of myosin II isoforms, can be used as an excitation–contraction uncoupler. METHODS: The specificity and potency of blebbistatin were examined by assaying the effects of blebbistatin on the contraction and basic cardiac electrophysiologic parameters of Langendorff-perfused rabbit hearts, isolated rabbit right ventricle and right atrium, and single rat ventricular myocytes using conventional ECG, surface electrograms, microelectrode recordings, and optical imaging with voltage-sensitive and Ca²⁺-sensitive dyes. Action potential morphology, ECG parameters, cardiac conduction, and refractoriness were determined after perfusion with 0.1–10 μM blebbistatin. RESULTS: Blebbistatin 5–10 μM completely eliminated contraction in all cardiac preparations but did not have any effect on electrical activity, including ECG parameters, atrial and ventricular effective refractory periods, and atrial and ventricular activation patterns. Blebbistatin 10 μM had no effects on action potential morphology in rabbit cardiac tissue. Blebbistatin inhibited single cellular contraction in a dose-dependent manner with half-maximal inhibitory concentration (IC₅₀) = 0.43 μM, without altering the morphologies of intracellular calcium transients. The blebbistatin effect was completely reversible by simultaneous washout and photobleaching by ultraviolet light CONCLUSION: Blebbistatin is a promising novel selective excitation–contraction uncoupler that can be used for optical imaging of cardiac tissues.