Intracellular Ca2+ is actively sequestered into the sarcoplasmic reticulum (SR), whereas the release of Ca2+ from the SR can be triggered by activation of the inositol 1,4,5-trisphosphate and ryanodine receptors. Uptake and release of Ca2+ across the SR membrane are electrogenic processes; accumulation of positive or negative charge across the SR membrane could electrostatically hinder the movement of Ca2+ into or out of the SR, respectively. We hypothesized that the movement of intracellular Cl− (Cl[Formula: see text]) across the SR membrane neutralizes the accumulation of charge that accompanies uptake and release of Ca2+. Thus inhibition of SR Cl− fluxes will reduce Ca2+ sequestration and agonist-induced release. The Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10−4 M), previously shown to inhibit SR Cl− channels, significantly reduced the magnitude of successive acetylcholine-induced contractions of airway smooth muscle (ASM), suggesting a “run down” of sequestered Ca2+ within the SR. Niflumic acid (10−4 M), a structurally different Cl− channel blocker, had no such effect. Furthermore, NPPB significantly reduced caffeine-induced contraction and increases in intracellular Ca2+ concentration ([Ca2+]i). Depletion of Cl[Formula: see text], accomplished by bathing ASM strips in Cl−-free buffer, significantly reduced the magnitude of successive acetylcholine-induced contractions. In addition, Cl− depletion significantly reduced caffeine-induced increases in [Ca2+]i. Together these data suggest a novel role for Cl[Formula: see text] fluxes in Ca2+ handling in smooth muscle. Because the release of sequestered Ca2+ is the predominate source of Ca2+ for contraction of ASM, targeting Cl[Formula: see text] fluxes may prove useful in the control of ASM hyperresponsiveness associated with asthma.