Exposure to ozone (O₃) induces airway hyperresponsiveness mediated partly through the release of substance P (SP) from nerve terminals in the airway wall. Although substantial evidence suggests that SP is released by sensory nerves, SP is also present in neurons of airway ganglia. The purpose of this study was to investigate the role of intrinsic airway neurons in O₃-enhanced airway responsiveness in ferret trachea. To remove the effects of sensory innervation, segments of ferret trachea were maintained in culture conditions for 24 h prior to in vitro exposed 2 ppm O₃ or air for 1 h. Sensory nerve depletion was confirmed by showing that capsaicin did not affect tracheal smooth muscle responsiveness to cholinergic agonist or contractility responses to electrical field stimulation (EFS). Contractions of isolated tracheal smooth muscle to EFS were significantly increased after in vitro O₃-exposure but constrictor response to cholinergic agonist was not altered. Pretreatment with CP-99994, an antagonist of neurokinin 1 (NK₁)-receptor, attenuated the increased contraction to EFS after O₃ exposure but had no effect in the air exposure group. The number of SP-positive neurons, in longitudinal trunk ganglia, the extent of SP innervation to superficial muscular plexus nerve cell bodies, and SP nerve fiber density in tracheal smooth muscle all increased significantly after O₃ exposure. The results show that release of SP from intrinsic airway neurons contributes to O₃-enhanced tracheal smooth muscle responsiveness by facilitating acetylcholine release from cholinergic nerve terminals.