Studies were performed to define the peptidergic nature of intramural nerves in the human esophagus. Cryosections of uninvolved surgically resected tissues from 14 individuals were studied by immunofluorescence for the localization of 10 neuropeptides. Myenteric neurons showed bombesin-, calcitonin gene-related peptide-, galanin-, substance P-, vasoactive intestinal polypeptide-, leucine-enkephalin-, methionine-enkephalin-, neuropeptide Y-, and somatostatin-like immunoreactivity. Submucous neurons had all the above except neuropeptide Y, methionine-enkephalin, leucine-enkephalin, and bombesin. Both groups of neurons received nerve terminations positive for calcitonin gene-related peptide, galanin, neuropeptide Y, substance P, and vasoactive intestinal polypeptide. Myenteric neurons additionally received terminations positive for neuropeptide Y, methionine-enkephalin, and somatostatin. All muscle layers had varicose fibers that reacted for calcitonin gene-related peptide, galanin, neuropeptide Y, and substance P. Longitudinal and circular muscle received few nerves reactive for leucine-enkephalin, whereas methionine-enkephalin was localized in a few nerve endings in the circular muscle. Somatostatin- and bombesin-reactive nerves occurred in longitudinal muscle. No cholecystokinin-reactive nerves were found. This study extends the results of previous studies and shows the previously undescribed presence of calcitonin gene-related peptide- and galanin-reactive nerves in the human esophagus and identifies neuropeptides that may serve as motor, sensory, and modulatory neurotransmitters of esophageal nerves.