Exposure to ozone (O₃) enhances airway responsiveness, which is mediated partly by the release of substance P (SP) from airway neurons. In this study, the role of intrinsic airway neurons in O₃-induced airway responses was examined. Ferrets were exposed to 2 ppm O₃ or air for 1 h. Reactivity of isolated tracheal smooth muscle to cholinergic agonists was significantly increased after O₃ exposure, as were contractions to electrical field stimulation at 10 Hz. Pretreatment with CP-99994, a neurokinin type 1 receptor antagonist, partially abolished the O₃-induced reactivity to cholinergic agonists and electrical field stimulation. The O₃-enhanced airway responses were present in tracheal segments cultured for 24 h, a procedure shown to deplete sensory nerves while maintaining viability of intrinsic airway neurons, and all the enhanced smooth muscle responses were also diminished by CP-99994. Immunocytochemistry showed that the percentage of SP-containing neurons in longitudinal trunk and the percentage of neurons innervated by SP-positive nerve fibers in superficial muscular plexus were significantly increased at 1 h after exposure to O₃. These results suggest that enhanced SP levels in airway ganglia contribute to O₃-induced airway hyperresponsiveness.