This study investigated the effects of elevated, physiological levels of intracellular free [Ca2+] on depolarization-induced force responses, and on passive and active force production by the contractile apparatus in mechanically skinned fibres of toad iliofibularis muscle. Excitation–contraction (EC) coupling was retained after skinning and force responses could be elicited by depolarization of the transverse-tubular (T-) system. Raising the cytoplasmic [Ca2+] to 1 μm or above for 3 min caused an irreversible reduction in the depolarization-induced force response by interrupting the coupling between the voltage sensors in the T-system and the Ca2+ release channels in the sarcoplasmic reticulum. This uncoupling showed a steep [Ca2+] dependency, with 50% uncoupling at 1.9 μm Ca2+. The uncoupling occurring with 2 μm Ca2+ was largely prevented by the calpain inhibitor leupeptin (1 mm). Raising the cytoplasmic [Ca2+] above 1 μm also caused an irreversible decline in passive force production in stretched skinned fibres in a manner graded by [Ca2+], though at a much slower relative rate than loss of coupling. The progressive loss of passive force could be rapidly stopped by lowering [Ca2+] to 10 nm, and was almost completely inhibited by 1 mm leupeptin but not by 10 μm calpastatin. Muscle homogenates preactivated by Ca2+ exposure also evidently contained a diffusible factor that caused damage to passive force production in a Ca2+-dependent manner. Western blotting showed that: (a) calpain-3 was present in the skinned fibres and was activated by the Ca2+exposure, and (b) the Ca2+ exposure in stretched skinned fibres resulted in proteolysis of titin. We conclude that the disruption of EC coupling occurring at elevated levels of [Ca2+] is likely to be caused at least in part by Ca2+-activated proteases, most likely by calpain-3, though a role of calpain-1 is not excluded.