Airway surface liquid (ASL) volume depletion and mucus accumulation occur in cystic fibrosis (CF). The ASL comprises a superficial mucus layer (ML) overlying a periciliary fluid layer (PCL) that contacts surface epithelial cells. We measured viscosity of the ML and PCL from the diffusion of FITC-dextran dissolved in the ASL of unperturbed, well-differentiated primary cultures of human bronchial epithelia grown at an air-liquid interface. Diffusion was measured by fluorescence recovery after photobleaching, using a perfluorocarbon immersion lens and confocal fluorescence detection. Bleaching of an in-plane 6-μm-wide region was done in which diffusion coefficients were computed using solution standards of specified viscosity and finite-element computations of 2-layer dye diffusion in 3 dimensions. We found remarkably elevated viscosity in both ML and PCL of CF vs. non-CF bronchial epithelial cell cultures. Relative viscosities (with saline= 1) were in the range 7–10 in the non-CF ML and PCL, and 25–30 in both ML and PCL in CF, and greatly reduced by amiloride treatment or mucin washout. These data indicate that the CF airway surface epithelium, even without hyperviscous secretions from submucosal glands, produces an intrinsically hyperviscous PCL and ML, which likely contributes to CF lung disease by impairment of mucociliary clearance. Our results challenge the view that the PCL is a relatively watery, nonviscous fluid layer in contact with a more viscous ML, and offer an explanation for CF lung disease in the gland-free lower airways.—Derichs, N., Jin, B.-J., Song, Y., Finkbeiner, WE, Verkman, AS Hyperviscous airway periciliary and mucous liquid layers in cystic fibrosis measured by confocal fluorescence photobleaching.