Increasing concentrations of MgADP- or MgCDP- in the millimolar range cause an increase in the maximum Ca2+ -activated tension that a skinned rabbit soleus muscle fiber can develop in the presence of 2 mM MgATP2- or MgCTP2- respectively. In contrast, the maximal Ca2+ -activated ATPase activity of the fiber decreases in the presence of MgADP-. As the nucleoside diphosphate (MgADP- or MgCDP-) concentration is increased, the Ca2+ concentration required for half-maximal activation of tension is reduced. MgADP- has a similar effect on the Ca2+ concentration required to half-maximally activate the fiber ATPase. The effects on tension are due to magnesium nucleoside diphosphate and not some other form of nucleoside diphosphate since the effects occur at both low (pMg 4) and control (pMg 3) Mg2+ concentrations. Cooperativity, as judged by the Hill "n" value relating isometric tension and Ca2+, is less in the presence of 5 mM MgADP- as compared to a control (no added MgADP-) "n" value. Increasing concentrations of inorganic phosphate (Pi) in the millimolar range decrease maximum Ca2+ -activated tension, and increase the concentration of Ca2+ required to half-maximally activate tension, effects opposite to those of MgADP. These data are consistent with the hypothesis that cooperative interactions between actin and myosin can affect the affinity of troponin for Ca2+.