We found previously that squid myosin B was markedly different from squid myosin in the KCl concentration dependence of Ca-ATPase activity, i.e. the maximal activity of squid myosin B was obtained at 0.2-0.3M KCl and a rapid decrease in the activity occurred as the KCl concentration decreased. In contrast, squid myosin showed a maximum activity at 0.1M KCl. A further study was conducted and the results obtained were as follows: 1) At KCl concentrations lower than 0.2M, the Ca-ATPase activity of squid myosin was remarkably decreased by addition of F-actin. 2) A maximum turbidity of acto-squid myosin was also obtained at 0.2M KCl. 3) A concomitant activation of Mg-ATPase of myosin occurred when the Ca-ATPase activity of squid myosin was inhibited by F-actin. 4) The reduced activity of Ca-ATPase of acto-squid myosin in the low KCl medium was fully recovered upon addition of 1M urea. 5) The Ca-ATPase activity of squid HMM was no longer inhibited by F-actin. It is therefore concluded that the inhibition of Ca-ATPase activity of squid myosin by F-actin occurred only when a thick filament of myosin was applied to ATPase assay.