Regulation by calcium and myosin-S1 of the acceleration of the rate of phosphate release from myosin-ADP-inorganic phosphate (M-ADP-Pi) by the thin filament actin-tropomyosin (Tm)-troponin (Tn), was measured directly by using double mixing stopped-flow experiments with fluorescent phosphate binding protein. At low calcium and without rigor myosin-S1, saturating concentrations of thin filaments accelerate the rate of phosphate dissociation from M-ADP-Pi 8-fold, from 0.08 to 0.64 s(-1). If either myosin-S1 or calcium is bound to the thin filaments, phosphate release is a biphasic process in which the fast phase is the dissociation of Pi from actoTmTnM-ADP-Pi and the slow phase is limited by the hydrolysis of actoTmTnM-ATP to actoTmTnM-ADP-Pi. The maximum accelerations of the fast components by saturating thin filaments (relative to M-ADP-Pi alone) are: approximately equal 200-fold, 16 s(-1) (calcium only); approximately equal 400-fold, 30 s(-1) (EGTA and rigor S1); and approximately equal1,000-fold, 75 s(-1) (calcium and rigor S1). The maximum rate of phosphate dissociation attained with S1 and calcium bound to the thin filament is the same as for unregulated actin. Regulation of the rate of phosphate dissociation by calcium and myosin-S1 is partially explained by the model of Geeves [McKillop, D. F. and Geeves, M. A. (1993) Biophys. J. 65, 693-701], in which calcium and rigor S1 perturb the equilibria among three states of the thin filament (blocked, closed, and open). However, a quantitative description of the regulatory mechanism requires acceleration by calcium of an additional step of the mechanism, either phosphate dissociation or a preceding conformational change.