Both the nodose and jugular vagal ganglia provide sensory innervation to the airways. The purpose of this study was to localize and characterize the substance P (SP) content of vagal afferent neurons that project specifically to the tracheal epithelium. A retrograde neuronal tracer, fast blue dye or rhodamine-labeled latex microspheres, was instilled into the guinea pig trachea. After 7 d, the nodose and jugular ganglia were removed, sectioned, and prepared for immunocytochemistry. Sections of tracheal mucosa demonstrated that fast blue dye diffused throughout the airway wall, whereas the rhodamine-labeled microspheres, as expected, did not penetrate the basement membrane and were thus localized to the epithelium. When the diffusible fast blue dye was used, approximately 60% of the labeled neurons were found in the nodose ganglia and 40% in the jugular ganglia. By contrast, when the beads were used to label only epithelial nerve fibers, 97 +/- 1% of the tracheal neurons taking up the dye were derived from jugular neurons, 60 +/- 6% of which contained SP immunoreactivity. These studies demonstrate that, in contrast to the submucosa, nerve fibers innervating the epithelium of the trachea are derived nearly exclusively from neurons with cell bodies in the jugular ganglia.