In skeletal muscle fibers, the coupling between excitation of the surface membrane and the release of Ca2+ from the sarcoplasmic reticulum is irreversibly disrupted if cytoplasmic Ca2+ concentration ([Ca2+]) is raised to micromolar levels for a prolonged period. This excitation-contraction (EC) uncoupling may contribute to muscle weakness after some types of exercise and in certain muscle diseases and has been linked to structural alteration of the triad junctions, but its molecular basis is unclear. Both μ-calpain, a ubiquitous Ca2+-activated protease, and muscle-specific calpain-3 become autolytically activated at micromolar Ca2+ and have been suggested to be responsible for the uncoupling. This study used controlled Ca2+ exposure in mechanically skinned fibers from extensor digitorum longus muscle to show that EC uncoupling still occurs in muscle fibers of calpain-3-deficient mice, with a Ca2+ dependence indistinguishable from that in normal mice and rats. Western blotting of muscle fibers that had been partially EC uncoupled by exposure to an intermediate Ca2+ level (∼5 μM Ca2+ for 3 min, no ATP) showed the presence of autolytic activation of a proportion of the μ-calpain present, but with little or no activation of calpain-3. Homogenates of normal and calpain-3-deficient muscles exposed to micromolar Ca2+ displayed similar levels of diffusible proteolytic activity, as gauged by the rate of decline of passive force in stretched, skinned muscle fibers. Exogenously added μ-calpain, preactivated by elevated [Ca2+] and applied in the presence of 1 μM Ca2+, disrupted EC coupling in a manner similar to raised [Ca2+]. We conclude that calpain-3 is not responsible for Ca2+-induced disruption of EC coupling, but that μ-calpain is a plausible candidate.