Agonist-induced contraction of airway smooth muscle (ASM) can be triggered by an elevation in the intracellular Ca2+ concentration, primarily through the release of Ca2+ from the sarcoplasmic reticulum (SR). The refilling of the SR is integral for subsequent contractions. It has been suggested that Ca2+ entry via store-operated cation (SOC) and receptor-operated cation channels may facilitate refilling of the SR. Indeed, depletion of the SR activates substantial inward SOC currents in ASM that are composed of both Ca2+ and Na+. Accumulation of Na+ within the cell may regulate Ca2+ handling in ASM by forcing the Na+/Ca2+ exchanger (NCX) into the reverse mode, leading to the influx of Ca2+ from the extracellular domain. Since depletion of the SR activates substantial inward Na+ current, it is conceivable that the reverse mode of the NCX may contribute to the intracellular Ca2+ pool from which the SR is refilled. Indeed, successive contractions of bovine ASM, evoked by various agonists (ACh, histamine, 5-HT, caffeine) were significantly reduced upon removal of extracellular Na+; whereas contractions evoked by KCl were unchanged by Na+ depletion. Ouabain, a selective inhibitor of the Na+/K+ pump, had no effect on the reductions observed under normal and zero-Na+ conditions. KB-R7943, a selective inhibitor of the reverse mode of the NCX, significantly reduced successive contractions induced by all agonists without altering KCl responses. Furthermore, KB-R7943 abolished successive caffeine-induced Ca2+ transients in single ASM cells. Together, these data suggest a role for the reverse mode of the NCX in refilling the SR in ASM following Ca2+ mobilization.