We investigated the regulatory roles of MgADP and free Ca2+ in isometric tension development in skinned bovine cardiac muscle. We found that, in the relaxed state without free Ca2+, MgADP elicited a sigmoidal increase in active tension, as is the case in skeletal muscle (ADP-contraction). The critical MgADP concentration, at which the tension increment became half-maximal, increased in proportion to MgATP concentration, with a slope of approximately 1 for cardiac and 4 for skeletal muscle. Raising the free Ca2+ concentration decreased the critical MgADP concentration in proportion to the free Ca2+ concentration. In addition, the apparent Ca2+ sensitivity of tension development increased with MgADP, while decreasing with inorganic phosphate (Pi); MgADP suppressed the Ca2+- desensitizing effect of Pi in a concentration-dependent manner. These activating effects of MgADP were quantitatively assessed by means of a model based upon the kinetic scheme of actomyosin ATPase. These experimental results and model simulation suggest that the state of thin filaments is synergistically regulated by both the binding of Ca2+ to troponin and the formation of the actomyosin–ADP complex.