The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters V max and K ATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in K ATPase value and to a lesser extent the decrease in V max, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of V max and K ATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.