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Current hypotheses describing the function of normal airway surface liquid (ASL) in lung defense are divergent. One theory predicts that normal airways regulate ASL volume by modulating the flow of isosmotic fluid across the epithelium, whereas an alternative theory predicts that ASL is normally hyposmotic. These hypotheses predict different values for the osmotic water permeability (P_f) of airway epithelia. We measured P_f of cultures of normal and cystic fibrosis (CF) airway epithelia that, like the native tissue, contain columnar cells facing the lumen and basal cells that face a basement membrane. Xz laser scanning confocal microscopy recorded changes in epithelial height and transepithelial volume flow in response to anisosmotic challenges. With luminal hyperosmotic challenges, transepithelial and apical membrane P_f are relatively high for both normal and CF airway epithelia, consistent with an isosmotic …