Blebbistatin is a small molecule inhibitor that selectively and strongly binds to myosin II, inhibiting its activity. The study reveals that blebbistatin binds to the ATPase intermediate of myosin II with ADP and phosphate bound at the active site, slowing down phosphate release. It does not interfere with myosin binding to actin or ATP-induced actomyosin dissociation, but instead blocks the myosin heads in a product complex with low actin affinity. Molecular docking simulations suggest that the productive binding site of blebbistatin is in an aqueous cavity between the nucleotide pocket and the actin-binding interface of the myosin head. This binding site is accessible in the apo and open states of myosin, but the switch II closed conformation may stabilize a more open state of the actin cleft, increasing blebbistatin affinity. Blebbistatin's binding is three times stronger in the closed state than in the open state, consistent with experimental data. The compound's high selectivity and affinity make it a valuable tool for studying myosin II function in muscle physiology and cell biology. Blebbistatin blocks myosin II in an actin-detached state, preventing rigid actomyosin cross-linking, which is advantageous in in vivo applications. The study also shows that blebbistatin does not significantly affect actin binding or ATP-induced actomyosin dissociation, indicating that it does not interfere with the communication between ATP- and actin-binding sites. These findings support the idea that blebbistatin is a uncompetitive inhibitor, binding to a nucleotide-bound enzyme intermediate with high affinity, and significantly lowering the Vmax value. Overall, blebbistatin is a useful compound for investigating myosin II motor activity due to its isoform specificity and ability to block myosin II in an actin-detached state.Blebbistatin is a small molecule inhibitor that selectively and strongly binds to myosin II, inhibiting its activity. The study reveals that blebbistatin binds to the ATPase intermediate of myosin II with ADP and phosphate bound at the active site, slowing down phosphate release. It does not interfere with myosin binding to actin or ATP-induced actomyosin dissociation, but instead blocks the myosin heads in a product complex with low actin affinity. Molecular docking simulations suggest that the productive binding site of blebbistatin is in an aqueous cavity between the nucleotide pocket and the actin-binding interface of the myosin head. This binding site is accessible in the apo and open states of myosin, but the switch II closed conformation may stabilize a more open state of the actin cleft, increasing blebbistatin affinity. Blebbistatin's binding is three times stronger in the closed state than in the open state, consistent with experimental data. The compound's high selectivity and affinity make it a valuable tool for studying myosin II function in muscle physiology and cell biology. Blebbistatin blocks myosin II in an actin-detached state, preventing rigid actomyosin cross-linking, which is advantageous in in vivo applications. The study also shows that blebbistatin does not significantly affect actin binding or ATP-induced actomyosin dissociation, indicating that it does not interfere with the communication between ATP- and actin-binding sites. These findings support the idea that blebbistatin is a uncompetitive inhibitor, binding to a nucleotide-bound enzyme intermediate with high affinity, and significantly lowering the Vmax value. Overall, blebbistatin is a useful compound for investigating myosin II motor activity due to its isoform specificity and ability to block myosin II in an actin-detached state.