In the present study, we describe a subset of nerve fibers, characterized by their immunoreactivity for the calcium-binding protein calretinin, that are densely and selectively associated with cholinergic postganglionic neurons in the guinea pig tracheal ganglia. Retrograde neuronal tracing with cholera toxin B, combined with immunohistochemical analyses, showed that these nerve fibers do not originate from sensory neurons in the nodose, jugular, or dorsal root ganglia or from motor neurons in the nucleus ambiguus, dorsal motor nucleus of the vagus nerve, spinal cord, stellate ganglia, or superior cervical ganglia. Calretinin-immunoreactive nerve fibers disappeared from tracheal segments after 48 h in organotypic culture, indicating that the fibers were of extrinsic origin. However, calretinin-positive nerve fibers persisted in tracheal ganglia when tracheae were cocultured with the adjacent esophagus intact. Immunohistochemical analysis of the esophagus revealed a population of cholinergic neurons in the esophageal myenteric plexus that coexpressed calretinin. In functional studies, electrical stimulation of the esophagus in vitro evoked measurable contractions of the trachea. These contractions were not altered by prior organotypic culture of the trachea and esophagus to remove the extrinsic innervation to the airways but were significantly (P < 0.05) inhibited by the ganglionic blocker hexamethonium or by physical disruption of the tissue connecting the trachea and esophagus. These data suggest that a subset of esophageal neurons, characterized by the expression of calretinin and acetylcholine, provide a previously unrecognized excitatory input to tracheal cholinergic ganglia in guinea pigs.