I n human cardiomyopathy, anatomical abnormalities such as hypertrophy and fibrosis contribute to the risk of ventricular arrhythmias and sudden death. Here we have shown that increased myofilament Ca²⁺ sensitivity, also a common feature in both inherited and acquired human cardiomyopathies, created arrhythmia susceptibility in mice, even in the absence of anatomical abnormalities. In mice expressing troponin T mutants that cause hypertrophic cardiomyopathy in humans, the risk of developing ventricular tachycardia was directly proportional to the degree of Ca²⁺ sensitization caused by the troponin T mutation. Arrhythmia susceptibility was reproduced with the Ca²⁺-sensitizing agent EMD 57033 and prevented by myofilament Ca²⁺ desensitization with blebbistatin. Ca²⁺ sensitization markedly changed the shape of ventricular action potentials, resulting in shorter effective refractory periods, greater beat-to-beat variability of action potential durations, and increased dispersion of ventricular conduction velocities at fast heart rates. Together these effects created an arrhythmogenic substrate. Thus, myofilament Ca²⁺ sensitization represents a heretofore unrecognized arrhythmia mechanism. The protective effect of blebbistatin provides what we believe to be the first direct evidence that reduction of Ca²⁺ sensitivity in myofilaments is antiarrhythmic and might be beneficial to individuals with hypertrophic cardiomyopathy.