The mechanism of ATP hydrolysis in myofibrils can be studied by following the time course of tryptophan fluorescence. Stoichiometric quantities of ATP produce an enhancement of the tryptophan fluorescence in stirred suspensions of rabbit psoas myofibrils at pCa greater than 7. Approximately 1 mol of ATP/myosin head is required to obtain the maximum fluorescence enhancement of 4-6%. Upon the addition of quantities of ATP greater than 1 mol/mol of myosin head, the fluorescence rapidly increases to a steady state, which lasts for a period that is proportional to the amount of ATP added. The fluorescence then decays to the initial level with a half-time of approximately 40 s at 20 degrees C. Hydrolysis of [gamma-32P]ATP at pCa greater than 7 in myofibrils has an initial burst of approximately 0.7 mol/mol of myosin head that is followed by a constant rate of hydrolysis. The duration of the steady state hydrolysis is identical to the duration of the enhancement of tryptophan fluorescence. A lower limit of 5 X 10(5) M-1 S-1 was obtained for the second order rate constant of the fluorescence enhancement by ATP. At pCa of 4, the duration of the fluorescence enhancement is one-tenth to one-twentieth as long as at pCa greater than 7; this is consistent with the increased steady state rate of ATP hydrolysis at higher calcium concentrations. The time course of the fluorescence enhancement observed in myofibrils during ATP hydrolysis is qualitatively and quantitatively similar to that observed with actomyosin-S1 in solution. These results suggest that the kinetic mechanism of ATP hydrolysis that has been well established by studies of actomyosin-S1 in solution also occurs in myofibrils.