Recent data indicate the clinical benefit of nebulized hypertonic saline in cystic fibrosis lung disease, with a proposed mechanism involving sustained increase in airway surface liquid volume. To account for the paradoxical observation that amiloride suppresses the beneficial effect of hypertonic saline, it has been previously concluded (Donaldson, S. H., Bennett, W. D., Zeman, K. L., Knowles, M. R., Tarran, R., and Boucher, R. C. (2006) N. Engl. J. Med. 354, 241-250) that amiloride-inhibitable aquaporin (AQP) water channels in airway epithelia modulate airway surface liquid volume. Here, we have characterized water permeability and amiloride effects in well differentiated, primary cultures of human airway epithelial cells, stably transfected Fisher rat thyroid epithelial cells expressing individual airway/lung AQPs, and perfused mouse lung. We found high transepithelial water permeability (P(f), 54 +/- 5 microm/s) in airway epithelial cells that was weakly temperature-dependent and inhibited by >90% by reduced pH in the basal membrane-facing solution. Reverse transcription-PCR and immunofluorescence suggested the involvement of AQPs 3, 4, and 5 in high airway water permeability. Experiments using several sensitive measurement methods indicated that amiloride does not inhibit water permeability in non-cystic fibrosis (non-CF) or CF airway epithelia, AQP-transfected Fisher rat thyroid cells, or intact lung. Our data provide evidence against the mechanism proposed by Donaldson et al. to account for the effects of amiloride and hypertonic saline in CF lung disease, indicating the need to identify alternate mechanisms.