Smooth muscle specimens from the lower esophageal sphincter (LES) region of pig and man were analyzed for vasoactive intestinal peptides (VIP), substance P (SP), enkephalin, neuropeptide Y (NPY), gastrin/cholecystokinin (CCK), neurotensin, and somatostatin using immunocytochemistry and radioimmunoassay. VIP-, SP-, enkephalin-, and NPY-immunoreactive nerve fibers were observed in the LES of both species, whereas nerve fibers containing gastrin/CCK, neurotensin, and somatostatin could not be demonstrated. The peptide-containing nerve fibers occurred in the intramural ganglia and in the smooth muscle layers. There was a rich supply of VIP- and NPY-immunoreactive fibers, whereas the supply of SP- and enkephalin-immunoreactive nerve fibers were moderate in number in both species examined. The concentration of VIP, SP, enkephalin, and NPY was comparable in the two species. The present study shows that the pattern of peptidergic innervation of the LES is similar in pig and man. It is proposed that neuronal VIP, SP, enkephalin, and NPY may serve to modulate the motor activity of the LES and that the pig is a suitable experimental animal for the study of regulatory peptides and LES functions.