In our model of regulation, the observed lack of cooperativity in the binding of myosin subfragment 1 (S-1) with bound ATP to the troponin-tropomyosin-actin complex (regulated actin) is explained by S-1.ATP having about the same affinity for the conformation of the regulated actin that activates the myosin ATPase activity (turned-on form) and the conformation that does not activate the myosin ATPase activity (turned-off form). This predicts that, in the absence of Ca2+, S-1.ATP should not turn on the regulated actin filament. In the present study, we tested this prediction by using either unmodified S-1 or S-1 chemically modified with N,N'-p-phenylenedimaleimide (pPDM X S-1) so that functionally it acts like S-1.ATP, although it does not hydrolyze ATP. We found that, in the absence of Ca2+, neither S-1.ATP nor pPDM X S-1.ATP significantly turns on the ATPase activity of the regulated complex of actin and S-1 (acto X S-1). In contrast, in the presence of Ca2+, pPDM X S-1.ATP binding almost completely turns on the regulated acto.S-1 ATPase activity. These results can be explained by our original cooperativity model, with pPDM X S-1.ATP binding only approximately equal to 2-fold more strongly to the turned-on form than to the turned-off form of regulated actin. However, our results are not consistent with our alternative model, which predicts that if pPDM X S-1.ATP binds to actin in the absence of Ca2+ but does not turn on the ATPase activity, then it should also not turn on the ATPase activity in the presence of Ca2+.