Previous work described in the rat a circumscribed, partly somatostatinergic, interneuronal projection from the esophageal afferent part of the nucleus of the solitary tract (NTSc) to esophageal motor neurons in the compact formation of the nucleus ambiguous (NAcf: Cunningham and Sawchenko, J Neurosci 9:1668, 1989). In the present study, axonal transport, immunohistochemical and in situ hybridization histochemical techniques were used to determine whether enkephalin (ENK), a peptide known to be expressed in a number of somatostatin-containing medullary cell groups, is also expressed in the projection from the NTSc to the NAcf. The results may be summarized as follows: 1) cells immunoreactive (IR) for prepro-enkephalin (ppENK)-derived peptides were found in the NTSc in colchicine-pretreated animals; in untreated animals, a dense ENK-IR terminal field was observed in the NAcf: sections stained with antisera against dynorphin-related peptides showed sparse staining in both regions; 2) signal indicating the presence of ppENK messenger RNA (mRNA) was found over the NTSc, including over a majority of cells identified using a retrograde tracing technique as projecting to the region of the NAcf; the signal for ppENK mRNA signal was greater than that for prepro-somatostatin (ppSS) in the NTSc; 3) a combined anterograde tracing-immunohistochemical technique demonstrated a strong correspondence between the distribution of inputs from the NTS to the NAcf, and the distribution of endogenous ENK-IR varicosities; in addition, leucine (L)-ENK-IR was found in an appreciable number of varicosities in the NAcf that had been anterogradely labeled from the NTSc; 4) unilateral electrolytic lesions of the rostromedial NTS, which included the central subnucleus, virtually eliminated ENK-IR in the ipsilateral NAcf, while staining on the contralateral side was unaffected. Taken together, these studies provide evidence that ppENK- and ppSS-derived peptides are expressed in the pathway from the NTSc to the NAcf, a pathway thought to play a role in the reflex control of esophageal peristalsis.