Vapors of butter flavor often contain diacetyl, an alpha-diketone that imparts the aroma and flavor of butter. Diacetyl causes epithelial necrosis and inflammation in rat and mouse airways. Diacetyl-exposed workers can develop fixed airway obstruction, but some exposed workers have symptoms of other conditions including self-reported wheezing. Pharmacokinetic modeling indicates that, at given vapor concentrations, the rat trachea receives comparable doses to human intrapulmonary airways following diacetyl inhalation. Substance P (SP) in sensory neurons plays a critical role in airway hyperresponsiveness and inflammation. Therefore, we investigated the hypothesis that diacetyl inhalation alters sensory innervation and SP production in airway epithelium. Neurons in jugular and nodose ganglia projecting to the airways were retrogradely labeled by tracheal instillation of rhodamine microspheres. Rats (6/group) were exposed to 0, 25, 249, or 346 ppm diacetyl for 6 h. One day post-exposure, ganglia and upper trachea were processed for SP immunofluorescence. SP immunoreactive sensory nerve fibers and cell bodies were measured by morphometry. Diacetyl inhalation caused a dose-dependent increase in the number of SP immunoreactive airway neurons in jugular ganglia from 3.26 ± 3.0 (control) to 14.70 ± 5.9, 22.70 ± 8.4, and 25.5 ± 6.6 at 25, 249 and 346 ppm, respectively. In trachea, after inhaling 346 ppm diacetyl, sensory nerve fiber density was unchanged in the fields with intact epithelium. However, the innervation was significantly increased in the intact epithelium adjacent to denuded foci (5.7 fold) and in foci with detaching epithelium (3.3 fold). These findings suggest that acute diacetyl inhalation causes dose-dependent SP production in airway neurons of jugular ganglia and increased epithelial innervation in tracheal foci with specific types of epithelial injury.