Osmotic water permeability (P _f ) was measured in spheroid-shaped human nasal airway epithelial explants pre-exposed to increasing levels of hyperosmotic stress. The fluid-filled spheroids, derived from nasal polyps, were lined by a single cell layer with the ciliated apical cell membrane facing the outside. The P _f was determined from diameter changes of the spheroids in response to changes in bathing medium osmolarity forth and back between 300 and 225 mOsm·l⁻¹. Continuous diameter measurements also allowed determination of spontaneous fluid absorption. Hyperosmotic pretreatment (increase from 300 up to 600 mOsm·l⁻¹) caused a time- and osmolarity-dependent increase (up to ∼1.5 times) in epithelial P _f which was of similar magnitude in cystic fibrosis (CF) and non-CF spheroids. The effect saturated at ∼450 mOsm·l⁻¹ and at ∼24 h. Expression of aquaporin-5 (AQP5), studied by immunofluorescence and confocal microscopy, showed an increase in parallel with the increase in P _f following hyperosmotic stress. The AQP5 was localized both in cytoplasmic vesicles and in apical cell membranes. Spontaneous fluid absorption rates were equal in CF and non-CF spheroids and were not significantly influenced by hyperosmotic stress. The results suggest that hyperosmotic stress is an important activator of AQP-5 in human airway epithelium, leading to significantly increased transepithelial water permeability.