The amount of PCMB-titratable sulfhydryl group of actin was in the range of 6.1 to 6.8 moles per 6.1×l04g. Two moles of them reacted instantaneously with PCMB and were titratable with NEM. By titration of these two sulfhydryl groups with the reagent the polymerization of G-actin was not inhibited and F-actin was not depolymerized. Remaining sulfhydryl groups reacted slowly with PCMB and did not with NEM. The polymerization of G-actin was inhibited and F-actin was depolymerized by the slow binding of PCMB to the sulfhydryl groups of the latter class. The binding of PCMB to these groups was retarded by the addition of ATP but not by the addition of calcium. Thc excess right-handed helical content of G-actin decreased gradually with time from 37 to 23.5 per cent on the addition of sufficient amount of PCMB. Similar changes were also observed on the addition of 2 moles PCMB per 6.1 × l04g. actin. The inhibition of polymerization by PCMB was accompanied by removal of the ATP bound to G-actin. The amount of the tight1y bound calcium was 1.5 moles per 6.1 × l04g. actin. 0.9 mole of them was released by the addition of large amount of PCMB. Even by the addition of 2 moles of PCMB per 6.1 × l04g. actin, the binding of calcium to actin became loose. On the basis of these results, an attempt was made to elucidate the mechanism of binding of ATP to actin.