The binding of myosin subfragment-1 (S-1) to the F-actin-troponin-tropomyosin complex (regulated F-actin) was examined in the presence of ADP (ionic strength, 0.23 M; 22 degrees C) by using the ultracentrifuge and S-1 blocked at SH1 with iodo[14C]acetamide. S-1 . ADP binds with positive cooperativity to regulated F-actin, both in the presence and absence of calcium; it binds independently to unregulated actin. With and without Ca2+ at very low levels of occupancy of the regulated actin by S-1 . ADP, S-1 . ADP binds to the regulated actin with less than 1% of the strength that it binds to unregulated actin, whereas at high levels of occupancy of the regulated actin by S-1 . ADP, S-1 . ADP binds about 3-fold more strongly to the regulated actin than it does to unregulated actin. The major difference between the results obtained in the presence and absence of Ca2+ with regulated actin is that, in the absence of Ca2+, the binding of S-1 . ADP remains weak until a higher free S-1 . ADP concentration is reached and the transition to strong binding is much more cooperative. These results are consistent with a model that is basically similar to the cooperative binding model of Hill[Hill, T.L. (1952) J. Chem. Phys. 20, 1259-1273] and of Monod et al. [Monod, J., Wyman, J. & Changeux, J. (1965) J. Mol. Biol. 12, 88-118]: The regulated actin filament can exist in two forms, a weak-binding and a strong-binding form; and Ca2+ and S-1 . ADP, acting as allosteric effectors, shift the equilibrium between the two forms.