The yield of S-l*** from tryptic digests of HMM was 65%. The s20, w value of S-l was 5.03 S at a concentration of 14.2 mg per ml. S-l decomposed into smaller components at a rate of about 7% of total material per day in neutral salt solution at 0°C. A quarter as much degradation occurred on addition of 0.07 M sucrose. The Ca2+-ATPase [EC 3. 6.1. 3] activity of S-l decreased in proportion to increase in the amount of smaller components. In 4.6–5 M guanidine-HCl and in alkaline (pH 11– 11.5) solution, S-l showed a broad sedimentation pattern with S20, w values of the peak of 1.1 and 1.5 S, respectively. The pattern of elution from Sephadex G-200 and G-100 showed that S-l was degraded into small components in 5M guanidine-HCl or at pH 11. The ratio of the ATPase activities of myosin (M.W. 4.8×105), HMM (3.4×105)and S-l (1.2 ×105) on a molar basis was 1: 1: 0.5. This ratio was independent of the modifiers used. When myosin was subjected to carboxamidomethylation under the conditions where one specific cysteine residue was completely modified with IAA, the Ca2+- and Mg2+-ATPase activities of myosin increased 11.7 and 9.7 fold, respectively, but the EDTA-ATPase activity decreased by 76%. HMM and S-l were prepared from this modified myosin. The extents of activation and inhibition of ATPase activities by IAA during their production of these fragments remained almost constant. The amount of the initial stoichiometric burst of Pi-liberation per mole of S-l was 0.55 to 0.6 mole, which was half of those of myosin and HMM. The rate of initial rapid Pi-liberation was independent of the ATP concentration when the latter was lower than 0.6 mole per mole of S-l, but at concentrations above this, it increased with increase in ATP concentration. The amount of initial rapid Pi-libera-tion increased linearly with the ATP concentration until the amount of added ATP reached about 0.6 mole per mole of S-l, and at higher ATP concentrations remained constant at this value. It was concluded that tryptic digestion of one mole of HMM produced two moles of S-l, one of which had the active sites of ATP hydrolysis both via phosphoryla-tion and by simple hydrolysis. The molecular mechanism of so-called dissociation of actomyosin by ATP was discussed, considering different functions of these two S-l portions in the myosin molecule. Our molecular mechanism of muscle contraction proposed previously was modified in consideration of the different functions of S-l