When muscle is elongated, there is a length dependence of twitch potentiation and an increased Ca²⁺ sensitivity of the myofilaments. Changes in the charge potential of myofilaments, induced by a decrease in pH, are known to abolish the length dependence of Ca²⁺ sensitivity. This study was aimed at testing the hypothesis that a decrease in pH, and the concomitant loss of length dependence of Ca²⁺ sensitivity, depresses the length dependence of staircase potentiation. In vitro, isometric twitch contractions of fiber bundles dissected from the mouse extensor digitorum longus, performed before and after 10 s of 10-Hz stimulation (i.e., the staircase potentiation protocol) were analyzed at five different lengths, ranging from optimal length for maximal force production (L_o; = 12 ± 0.7 mm) to L_o + 1.2 mm (L_o + 10%). These measurements were made at an extracellular pH of 6.6, 7.4, and 7.8 (pH changes induced by altering the CO₂ concentration of the bath solution). At pH 7.4 and 7.8, the degree of potentiation after 10-Hz stimulation showed a linear decrease with increased fiber bundle length (r² = 0.95 and r² = 0.99, respectively). At pH 6.6, the length dependence of potentiation was abolished, and the slope of the length-potentiation relationship was not different from zero (r² = 0.05). The results of this study indicate that length dependence of potentiation in intact skeletal muscle is abolished by lowering the pH. Because decreasing the pH decreases Ca²⁺ sensitivity and changes the charge potential of the filaments, the mechanism of length-dependent potentiation may be closely related to the length dependence of Ca²⁺ sensitivity, and changes in the charge potential of the myofilaments may be important in regulating this relationship.