Muscle contraction is driven by the relative sliding of actin- and myosin-containing filaments. This process can be reconstituted in in vitro motility assays, where fluorescent actin filaments travel over a lawn of myosin heads. The velocity of the traveling actin filaments has been shown to depend strongly on the free adenosine triphosphate (ATP) concentration, but below 2–4 μM ATP, filament movement is consistently abolished. Here we report that after a brief exposure of actomyosin to 1 mM ATP, actin filament motility persists down to nanomolar ATP concentrations.