The aim of the present study was to investigate the role of the autonomic nervous system in the regulation of airway epithelial ion transport in vivo. Rabbits were anaesthetized and mechanically-ventilated through a cannula inserted above the carina. The upper tracheal mucosa was exposed, and the electrical potential difference (PD) between the mucosal surface and the submucosal space was continuously measured by a high-impedance voltmeter under open-circuit conditions. Perfusion of the mucosa with atropine caused a rapid decline in PD from -20.1+/-2.0 to -15.2+/-0.9 mV (p<0.01), whereas phentolamine, propranolol, or the tachykinin antagonist, FK224, had no effect. Cutting both cervical vagus nerves decreased PD to the same degree as did atropine. Exogenously applied acetylcholine increased PD in a dose-dependent manner. Topical application of ipratropium bromide reduced the baseline value PD in a dose-dependent manner. The maximal decrease in PD was 43 +/- 0.3 mV (p<0.01), and the dose required to produce a half-maximal effect was 34 microg. Perfusion with either amiloride, a Na channel blocker, and diphenylamine-2-carboxylate, a Cl channel blocker, decreased the baseline PD, and the subsequent application of ipratropium bromide further decreased the PD in each case. We conclude that a cholinergic neural component may play a role in the generation of tracheal potential difference in vivo, probably involving stimulation by endogenously released acetylcholine of both Cl secretion and Na absorption across the airway epithelium.