Under ultrasonic vibration the bound Ca of F-actin exchanges with free Ca. The exchange of the F-actin-bound Ca is more rapid than the ATP hydrolysis catalyzed by F-actin under the same conditions. In the presence of ATP the bound Ca of F-actin cannot be removed by polyethylene sulfonate or urea on ultrasonic vibration. It can be removed, however, by EDTA or ethylene glycol bis( β-amino-ethyl ether)- N- N′-tetraacetic acid without inhibition of the ATPase activity of F-actin. The F-actin-bound Ca can also be removed by mercurials with concomitant inhibition of the F-actin ATPase. A relationship appears between ATPase activity of F-actin and those -SH groups which seem necessary for polymerization. The Ca-free F-actin, prepared by ultrasonic vibration in the presence of EDTA and ATP, maintains its viscosity and its ability to interact with L-myosin both at high and low ionic strength; however, it loses its ability to be depolymerized reversibly. The ATPase activity of actomyosin prepared from Ca-free F-actin can be inhibited by EDTA both in the presence and absence of Mg 2+.