Blebbistatin (BLEB) is a recently discovered compound that inhibits myosin-II ATPase activity. In this study, we tested BLEB in intact and skinned isolated rat cardiac trabeculae, rat intact myocytes, and single rabbit psoas myofibrils. BLEB (10 muM) reduced twitch force and cell shortening that was reversed by exposure to light. BLEB treatment of skinned trabeculae in the dark (1 hr) reduced Ca(2+)-activated force (EC(50) = 0.38 +/- 0.03 muM). Rapid (<5 ms) BLEB application in Ca(2+)-activated rabbit myofibrils reduced force proportional to [BLEB]. Two-photon Indo1-AM ratio-metric confocal line-scan microscopy revealed no impact of BLEB on the cytosolic Ca(2+) transient. BLEB reduced contractile force in skinned trabeculae without affecting tension-dependent myofilament ATPase activity. We conclude that BLEB specifically uncouples cardiac myofilament activation from Ca(2+) activation without affecting EC coupling or cross-bridge cycling parameters. This agent could be useful to uncouple myofilament contractility from electrical events that lead to sarcoplasmic reticulum Ca(2+) release in the cardiac myocyte (uncoupling agent) However, the compound is very sensitive to light, a property that limits its application to mechanistic physiological studies.