Cystic fibrosis (CF) airway epithelia are characterized by enhanced Na⁺ absorption probably due to a lack of downregulation of epithelial Na⁺ channels by mutant CF transmembrane conductance regulator. Extracellular nucleotides adenosine 5 ′ -triphosphate (ATP) and uridine 5 ′ -triphosphate (UTP) have been shown to activate alternative Ca^{2 +}-dependent Cl⁻ channels in normal and CF respiratory epithelia. Recent studies suggest additional modulation of Na⁺ absorption by extracellular nucleotides. In this study we examined the role of mucosal ATP and UTP in regulating Na⁺ transport in native human upper airway tissues from patients with 16 patients with CF and 32 non-CF control subjects. To that end, transepithelial voltage and equivalent short-circuit current (I _sc ) were assessed by means of a perfused micro-Ussing chamber. Mucosal ATP and UTP caused an initial increase in lumen-negative I _sc that was followed by a sustained decrease of I_sc in both non-CF and CF tissues. The amiloride-sensitive portion of I _sc was inhibited significantly in normal and CF tissues in the presence of either ATP or UTP. Both basal Na⁺ transport and nucleotide-dependent inhibition of amiloride-sensitive I _sc were significantly enhanced in CF airways compared with non-CF. Nucleotide-mediated inhibition of Na⁺ absorption was attenuated by pretreatment with the Ca^{2 +}–adenosine triphosphatase inhibitor cyclopiazonic acid but not by inhibition of protein kinase C with bisindolylmaleimide. These data demonstrate sustained inhibition of Na⁺ transport in non-CF and CF airways by mucosal ATP and UTP and suggest that this effect is mediated by an increase of intracellular Ca^{2 +}. Because ATP and UTP inhibit Na⁺ absorption and stimulate Cl⁻ secretion simultaneously, extracellular nucleotides could have a dual therapeutic effect, counteracting the ion transport defect in CF lung disease.