The regulation by calcium and rigor bound myosin-S1 of the acceleration of the rate of mdADP release from M-mdADP-Pi by skeletal muscle thin filaments (reconstituted from actin-tropomyosin-troponin), was measured using double mixing stopped-flow fluorescence with the nucleotide substrate 3'methylphenanthranoyl-2'deoxyATP (mdATP). The predominant mechanism of regulation is the acceleration of product dissociation by a factor of ~200 by thin filaments in the fully activated conformation (bound calcium and rigor S1) relative to the inhibited conformation (no bound calcium or rigor S1). In contrast, only two to three fold regulation is due to a change in actin affinity such as would be expected by "steric blocking" of the myosin binding site of the thin filament by tropomyosin. The binding of one ligand (either calcium or rigor-S1) produces partial activation of the rate of product dissociation but the binding of both is required to maximally accelerate product dissociation to a rate similar to that obtained with f-actin in the absence of regulatory proteins. The data support an allosteric regulation model in which the binding of either calcium or rigor S1 alone to the thin filament shift the equilibrium in favor of the active conformation but full activation requires binding of both ligands.