The steric blocking model of troponin-tropomyosin action suggests that relaxation of skeletal muscle is caused by tropomyosin physically preventing the binding of the myosin cross-bridge to actin in the absence of Ca2 plus. We tested this model by measuring the binding of myosin subfragment-one (S-1) to actin and regulated actin (actin plus troponin plus tropomyosin) with and without Ca2 plus in the presence of ATP. Stopped-flow turbidity measurements were used to determine the amount of S-1 binding to actin. At 25 degrees C, micron equals 17.5 mM the binding constant of S-1 to actin or regulated actin in the presence of Ca2 plus is 2.3 times 10 to the 4th power M-1. In the absence of Ca2 plus, when the ATPase is 94 percent inhibited, the binding of S-1 to regulated actin is reduced only two-fold (K equals 1.3 times 10 to the 4th power M-1). These data show that the inhibitory effect of troponin-tropomyosin on the actin activated S-1 ATPase without Ca2 plus is not due to inhibition of binding of S-1 to the actin filament in the presence of ATP. Rather, these data suggest that troponin-tropomyosin acts by blocking the release of Pi from the actin-S-1-ADP-Pi complex. In vivo this may correspond to blocking the rotation of the actin bound cross-bridge from the 90 degrees attached state to the 45 degrees attached state.