Agonist-induced contraction of airway smooth muscle (ASM) can be triggered by an elevation in the intracellular Ca(2+) concentration, primarily through the release of Ca(2+) from the sarcoplasmic reticulum (SR). The refilling of the SR is integral for subsequent contractions. It has been suggested that Ca(2+) 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 Ca(2+) and Na(+). Accumulation of Na(+) within the cell may regulate Ca(2+) handling in ASM by forcing the Na(+)/Ca(2+) exchanger (NCX) into the reverse mode, leading to the influx of Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) mobilization.