A nonlinear least squares analysis of Scatchard plots of Ca++ binding to troponin, native tropomyosin, and myosin B demonstrates that troponin does not possess two classes of independent binding sites. Since the data cannot be accounted for by assuming more than two classes of independent sites, we conclude that binding of one Ca decreases the affinity of troponin for binding the next. Negative interaction is increased in the presence of tropomyosin and further increased by actomyosin. The maximal Ca++ affinity of troponin at low fractional occupancy is not affected by tropomyosin but may be significantly reduced by actomyosin. Because of negative interaction, troponin's Ca++ affinity should vary during the course of contraction. This variation could profoundly affect translocations of Ca between troponin and reticulum and the time course of the active state.