Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%. Inhibitory caldesmon concentrations had little effect upon the rate of S1 binding to actin, actin-S1 dissociation by ATP, and dissociation of ADP from actin-S1⅐ADP; however the rate of phosphate release from the actin-S1⅐ADP⅐P i complex was decreased by more than 80%. In addition the transient of phosphate release displayed a lag of up to 200 ms. The presence of a lag phase indicates that a step on the pathway prior to phosphate release has become rate-limiting. Premixing the actin-tropomyosin filaments with myosin heads resulted in the disappearance of the lag phase. We conclude that caldesmon inhibition of the rate of phosphate release is caused by the thin filament being switched by caldesmon to an inactive state. The active and inactive states correspond to the open and closed states observed in skeletal muscle thin filaments with no evidence for the existence of a third, blocked state. Taken together these data suggest that at physiological concentrations, caldesmon controls the isomerization of the weak binding complex to the strong binding complex, and this causes the inhibition of the rate of phosphate release. This inhibition is sufficient to account for the inhibition of the steady state actomyosin ATPase by caldesmon and tropomyosin.Smooth muscle contractility and actomyosin ATP hydrolysis are regulated by Ca 2ϩ at two levels. The first involves phosphorylation of the myosin light chain by myosin light chain kinase upon stimulation by Ca 2ϩ -calmodulin. The second occurs via the thin filament-associated protein caldesmon. Caldesmon (CaD) 2 binds to actin, calmodulin, and tropomyosin (1) and inhibits the actin activation of both smooth and skeletal muscle myosin ATPase in vitro (2) and force production when added to smooth and skeletal muscle fibers (3-5). Caldesmon also regulates the actin activation of myosin I ATPase in smooth muscle (6). The smooth muscle thin filament is Ca 2ϩ -regulated and is made up of actin, tropomyosin, caldesmon, and a Ca 2ϩ -binding protein in molar ratios ϳ14:2:1:1 (7). Previous studies of the molecular mechanism of caldesmon inhibition of the actomyosin ATPase have focused on its effect on the ATPase steady state parameters K m and V max (8) and on the equilibrium binding of myosin to actin filaments (9 -12). A detailed mechanism by which caldesmon regulates the actin activation of myosin ATPase and force production has not been demonstrated by such experiments but two potentially conflicting models have been developed.A competition model proposes that ATPase inhibition by caldesmon is caused by a reduction in the formation of the weakly bound actomyosin complex as a...