Vertebrate skeletal muscle contraction is the result of a cyclic interaction of thick myosin filaments with the thin filaments, composed primarily of actin, troponin, and tropomyosin, causing these two sets of filaments to slide past each other (2, 3). This cycling is driven by the hydrolysis of ATP by myosin in a reaction which is activated by actin. When the sarcoplasmic reticulum lowers the free Ca2+ concentration from 10−5 to <10−7 m, muscle contraction ceases and the associated actin-activated myosin ATPase activity is inhibited. The proteins troponin and tropomyosin are responsible for this effect of Ca2+ on the interaction between myosin and actin (4, 5). The tropomyosin molecules lie end to end along the two grooves of the F-actin filament with each tropomyosin molecule binding to seven actin monomers (6). Troponin consists of three subunits, and one troponin molecule is bound to each tropomyosin molecule. The binding of Ca2+ to troponin determines the position of tropomyosin on the F-actin filament. At levels of Ca2+ low enough to cause relaxation, tropomyosin is positioned away from the central groove of the F-actin filament where it appears that it might interfere with the binding of the myosin cross-bridge (7–9). This structural work formed the basis of the steric blocking hypothesis which suggests that relaxation occurs when tropomyosin, in the “relaxed” position, physically blocks the binding of the myosin cross-bridge to actin. Three-dimensional reconstructions from electron micro-graphs have suggested that the myosin cross-bridge and the tropomyosin molecule may be in close contact with each other on the actin filament, a requirement for a steric blocking type model (10, 11). The steric blocking model predicts that in the absence of Ca2+ the degree of association of S-11 should be much weaker with regulated actin than with unregulated actin. In fact, Greene and Eisenberg (12) have recently demonstrated that, in the absence of Ca2+, the binding of S-1·ADP to regulated actin is strongly inhibited in a cooperative manner. At low levels of saturation of the actin filament with S-1·ADP, the binding of S-1·ADP to the regulated actin filament is about 103 weaker than at high levels of saturation. However, the fact that S-1·ADP binds weakly to regulated actin does not prove the steric blocking model since, in relaxed muscle, the cross-bridges normally exist with bound ATP (or ADP · Pi) and not bound ADP (13,14). Therefore, the steric blocking model predicts that troponin-tropomyosin should inhibit the binding of S-1·ADP·Pi as well as S-1·ADP to regulated actin in the absence of Ca2+. Using stopped flow turbidity measurements, we have previously measured the effect of Ca2+ on the association of S-1 · ATP and S-1 · ADP · Pi with regulated actin (15). Surprisingly, in the absence of Ca2+, the binding constant of S-1 · ATP or S-1 · ADP · Pi to regulated actin was only decreased to 56% of the value in the presence of Ca2+ although the rate of ATP hydolysis under the same conditions was decreased to 6% of the rate with Ca2+ present. These data suggest, in disagreement with the steric blocking model, that inhibition of the rate of ATP hydrolysis, in the absence of Ca2+, is not the result of inhibition of the binding of S-1 to regulated actin. In the present study, we have reinvestigated this problem using a different and more direct measurement of binding. Free S-1 was separated from actin-bound S-1 in the presence of ATP by rapid centrifugation in an air-driven ultracentrifuge and the free S-1 concentration was then determined by an ATPase assay. As in our earlier turbidity studies, we find very little effect of troponin-tropomyosin on the binding of S-1 to regulated actin in the presence of ATP. Similar confirmation of our turbidity results has already been reported by Wagner and Giniger (16). In the present study, we have also measured the binding at a higher ionic strength (50 mm) and here too have found no correlation between the inhibition of ATPase rate and the binding of S-1·ATP or S-1 · ADP · Pi to regulated actin. Finally, we demonstrate that the removal of Ca2+ affects the rate of regulated actin-activated S-1-ATPase activity primarily by lowering the maximum ATPase rate (Vmax) rather than the apparent binding constant of S-1 to actin (KATPase). These data imply that, in the absence of Ca2+, troponin-tropomyosin inhibits the ATPase activity by inhibiting a kinetic step in the cycle of ATP hydrolysis, perhaps Pi release.