We investigated the mechanism of the decreased myofilament Ca²⁺ responsiveness in stunned myocardium. The steady state force-[Ca²⁺] relationship was measured before and after skinning in thin ventricular trabeculae from control or stunned (20 minutes of ischemia, 20 minutes of reperfusion) rat hearts. [Ca²⁺]_i was determined using microinjected fura 2 salt in intact muscles, whereas the myofilaments of chemically skinned trabeculae were activated directly with solutions of varied [Ca²⁺]. Maximal Ca²⁺-activated force (F_max) before and after skinning was identical within either the control or stunned groups but was markedly depressed in both groups of stunned trabeculae (P<.001). After ischemia and reperfusion, the [Ca²⁺] required for 50% of maximal activation (Ca₅₀) was increased in both intact (control, 0.60±0.09 μmol/L; stunned, 0.85±0.09 μmol/L; P<.001) and skinned (control, 1.13±0.24 μmol/L; stunned, 1.39±0.21 μmol/L; P=.0025) trabeculae. These data indicate that the decreased Ca²⁺ responsiveness of stunned myocardium is due to intrinsic alterations of the myofilaments. Therefore, we tested the hypothesis that activation of proteases by reperfusion-induced Ca²⁺ overload decreases the Ca²⁺ responsiveness of the cardiac myofilaments. Force-[Ca²⁺] relations were compared before and 5 to 30 minutes after direct exposure of skinned trabeculae to calpain I (18 μg/mL, 20 minutes at [Ca²⁺]=10.8 μmol/L), a Ca²⁺-activated protease that is present in myocardium. Calpain I reduced F_max from 94.3±8.3 to 56±8.5 mN/mm² while increasing Ca₅₀ from 0.94±0.11 to 1.36±0.21 μmol/L (P<.01). Calpastatin, a specific calpain inhibitor, prevented the effects of calpain I on skinned trabeculae. The results show that the reduced Ca²⁺ responsiveness of stunned myocardium reflects alteration of the myofilaments themselves, not of soluble cytosolic factors, which can be faithfully reproduced by exposure to Ca²⁺-dependent protease.