Inhalation of nonisotonic solutions can elicit pulmonary obstruction in asthmatic airways. We evaluated the hypothesis that the respiratory epithelium is involved in responses of the airways to nonisotonic solutions using the guinea pig isolated, perfused trachea preparation to restrict applied agents to the mucosal (intraluminal) or serosal (extraluminal) surface of the airway. In methacholine-contracted tracheae, intraluminally applied NaCl or KCl equipotently caused relaxation that was unaffected by the cyclo-oxygenase inhibitor, indomethacin, but was attenuated by removal of the epithelium and Na+ and Cl- channel blockers. Na+-K+-2Cl- cotransporter and nitric oxide synthase blockers caused a slight inhibition of relaxation, whereas Na+,K+-pump inhibition produced a small potentiation. Intraluminal hyperosmolar KCl and NaCl inhibited contractions in response to intra- or extraluminally applied methacholine, as well as neurogenic cholinergic contractions elicited with electric field stimulation (+/- indomethacin). Extraluminally applied NaCl and KCl elicited epithelium-dependent relaxation (which for KCl was followed by contraction). In contrast to the effects of hyperosmolarity, intraluminal hypo-osmolarity caused papaverine-inhibitable contractions (+/-epithelium). These findings suggest that the epithelium is an osmotic sensor which, through the release of epithelium-derived relaxing factor, can regulate airway diameter by modulating smooth muscle responsiveness and excitatory neurotransmission.