The airway surface liquid (ASL) is the thin fluid layer lining the airways whose depth may be reduced in cystic fibrosis. Prior measurements of ASL depth have been made in airway epithelial cell cultures. Here, we established methodology to measure ASL depth to ~1-µm accuracy in ex vivo fragments of freshly obtained human and pig tracheas. Airway fragments were mounted in chambers designed for perfusion of the basal surface, and observation of the apical, fluorescently stained ASL by scanning confocal microscopy using a high numerical aperture lens immersed in perfluorocarbon. Measurement accuracy was verified using standards of specified fluid thickness. ASL depth in well-differentiated primary cultures of human nasal respiratory epithelium was 8.0 ± 0.5 µm (S.E., 10 cultures) under basal conditions, 8.4 ± 0.4 µm following ENaC inhibition by amiloride, and 14.5 ± 1.2 µm following CFTR stimulation by cAMP agonists. ASL depth in human trachea was 7.0 ± 0.7 µm under basal conditions, 11.0 ± 1.7 µm following amiloride, 17.0 ± 3.4 µm following cAMP agonists, and 7.1 ± 0.5 µm after CFTR inhibition. Similar results were found in pig trachea. This study provides the first direct measurements of ASL depth in intact human airways, and indicates the involvement of ENaC sodium channels and CFTR chloride channels in determining ASL depth. We suggest that CF lung disease may be caused by the inability of CFTR-deficient airways to increase their ASL depth transiently following secretory stimuli that in non-CF airways produce transient increases in ASL depth.