The effects of shortening distance at V_u, the unloaded shortening speed, and filament overlap on the amount of extra Ca²⁺ released during relaxation in muscle, as indicated by the bump area, were studied. Single, intact frog skeletal muscle fibers at 3°C were used. The myoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) was estimated by using fura 2 salt injected into the myoplasm. Ramps were applied, either at full overlap with different sizes or at varying overlaps with a fixed size, in the linear phase of relaxation. At full overlap, a plot of bump area vs. ramp size was fit by using a sigmoidal curve with one-half of the bump area equal to 25.9 nm. With a fixed ramp size of 100 nm/half-sarcomere, the plot of bump area vs. mean sarcomere length (SL_m) was fit by a straight line intersecting the SL_m axis at ~3.5 µm, close to just no overlap. The results suggest that the transition in the distribution of attached cross bridges from the isometric case to one appropriate for unloaded shortening at V_u is completed within 50 nm/half-sarcomere and support the view that attached cross bridges in the overlap zone influence the affinity of Ca²⁺ for troponin C in the thin filament.