The effects of dissociation of force-generating cross bridges on intracellular Ca²⁺, pCa-force, and pCa-ATPase relationships were investigated in mouse skeletal muscle. Mechanical length perturbations were used to dissociate force-generating cross bridges in either intact or skinned fibers. In intact muscle, an impulse stretch or release, a continuous length vibration, a nonoverlap stretch, or an unloaded shortening during a twitch caused a transient increase in intracellular Ca²⁺ compared with that in isometric controls and resulted in deactivation of the muscle. In skinned fibers, sinusoidal length vibrations shifted pCa-force and pCa-actomyosin ATPase rate relationships to higher Ca²⁺ concentrations and caused actomyosin ATPase rate to decrease at submaximal Ca²⁺ and increase at maximal Ca²⁺ activation. These results suggest that dissociation of force-generating cross bridges during a twitch causes the off rate of Ca²⁺ from troponin C to increase (a decrease in the Ca²⁺ affinity of troponin C), thus decreasing the Ca²⁺ sensitivity and resulting in the deactivation of the muscle. The results also suggest that the Fenn effect only exists at maximal but not submaximal force-activating Ca²⁺ concentrations.