The presteady state and steady state release of protons during hydrolysis of ATP by myosin has been studied by using a proteolytic subfragment of myosin, heavy meromyosin, isolated from rabbit skeletal muscle. Under the conditions of 0.5 M KCI, 9 mM MgCI2, pH 8, and 250, the rate of the H+ initial burst is proportional to the concentration of ATP added, and shows no plateau even when the concentration of ATP added is increased up to 750 ,AM. The apparent second order rate constant is approximately 7 X 105 M-1 sec1. When the concentration of ATP added is equal to or less than the concentration of heavy meromyosin catalytic sites, the H+ initial burst is followed by a slow exponential release of H+ with a rate constant of 0.017 sec'. This rate constant is equal to the steady state rate constant calculated for each heavy meromyosin catalytic site, suggesting that both sites split ATP independently. The magnitude of the H+ released per ATP bound is always 0.4 in the initial burst, and 0.6 in the slow exponential phase, which equals to a total of 1 H+ released per ATP hydrolyzed. On the basis of these observations, it is concluded that the H+ initial burst may well represent a fast ionization of H+ due to a conformational change as a result of nucleotide binding.It is now generally accepted that when ATP is added to rabbit skeletal myosin, heavy meromyosin (HMM), or subfragment-1 S-1, in the presence of excess Mg++, an initial rapid release of phosphate is observed prior to the onset of the steady state rate of phosphate release (1-3). Several workers have suggested that this initial phosphate burst might be correlated with the formation of an intermediate involved in the mechanism of muscle contraction in vivo (4-7). The rate of the initial phosphate burst has generally been reported to plateau at a rate of approximately 100 sec , but there is disagreement on the magnitude of the burst, with data varying from I to 2 mole of Pi released per mole of myosin or HMM (3,8,9 Eisenberg and Moos (17). The HMM was dialyzed against 0.5 M KCl before use. The stopflow experiments were performed using a two-syringe Aminco-Morrow stopflow mixer equipped with a 1-cm light path flow cell thermostated at 250C. The exhaust of the mixing chamber was slightly enlarged to reduce the flow resistance, which reduced the dead time of the instrument to approxiimately 2.1 msec for a reacting system containing 0.6-0.7 mg/ml of HMIM solution operating at 60 psi sampling pressure. The photodetection system was designed and built by Dr. P. 13. Chock and Mr. Ml.Greifner here at the National Institutes of Health. A detailed description of it will be published elsewhere. The time course