The effect of twitchin, a thick filament protein of molluscan muscles, on actin-myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using fluorescent probes specifically bound to Cys707 of myosin subfragment-1 and Cys374 of actin incorporated into ghost muscle fibers. The multi-step changes in mobility and spatial arrangement of myosin SH1 helix and actin subdomain-1 during the ATPase cycle have been revealed. For the first time, the inhibition of movement of myosin SH1 helix and actin subdomain-1 during the ATPase cycle and the decrease in the myosin head and actin affinity in the presence of unphosphorylated twitchin have been demonstrated. Phosphorylation of twitchin by the catalytic subunit of protein kinase A reversed this effect. These data imply a novel property of twitchin consisting in its ability to regulate in a phosphorylation-dependent manner the actin-myosin interaction during the ATPase cycle by the inhibition of transformation of the weak-binding actomyosin states into the strong-binding ones.