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Muscle contraction is regulated via two processes that both contribute to activation of the thin filament (actinþtroponinþtropomyosin). First, calcium (Caþþ) displaces tropomyosin, which increases the availability of myosin binding sites on actin. Second, myosin binding accelerates the attachment of neighboring myosin molecules to the thin filament. The relative importance and underlying mechanisms of these two processes remain unclear, therefore we devised assays to separately quantify their contribution. Myosin binding only activates nearby myosins, therefore by varying myosin density we varied the contribution of this process. Myosin-induced activation was eliminated by using a single myosin molecule in the laser trap assay to directly measure Caþþ's effect on myosin's interaction with a thin filament. Slightly increasing the myosin density generated mini ensembles of myosin that experienced weak coupling. Increasing the myosin density further using a motility assay created large myosin ensembles with strong myosin-induced activation, and thin filament speed was measured at increasing ATP and Caþþ levels. The data were analyzed with a mathematical model that de-convolves the effects of Caþþ-induced and myosin-induced activation. This analysis revealed (1) how Caþþ affects myosin's attachment to a thin filament; and (2) that myosin molecules are coupled over a Caþþ-independent distance L=400nm. Scaling up the model generated accurate predictions of isometric force production and Caþþ binding to the thin filament previously observed in muscle fibers, providing a molecular basis for how Caþþ and myosin contribute to filament activation.