The purpose of the present investigation was to identify and compare cholinergic intramural neurons in the lower esophageal sphincter and esophageal body by histochemical staining for acetylcholinesterase and the enzyme that synthesizes acetylcholine, choline acetyltransferase. Opossums were anesthetized and their abdominal cavity was opened by a midline incision to expose the esophagogastric junction. The lower esophageal sphincter was identified manometerically and localized in situ with markers. Tissues were removed, rapidly frozen in freon cooled with liquid nitrogen and serial cryostat sections were obtained from the lower esophageal sphincter and esophageal body. Sections were stained with one of the above histochemical procedures and adjacent sections were stained with Solachrome cyanin , which differentially stains nerve elements from muscle fibers. The muscle of the lower esophageal sphincter and esophageal body was stained with nonspecific cholinesterase with some selectivity of intensity of reaction in the various smooth muscle layers. All identifiable plexus neurons in the esophagus stained for nonspecific cholinesterase and acetylcholinesterase. Nerve fiber tracts were also stained for acetylcholinesterase within the longitudinal and circular layers of the tunica muscularis. Reaction for choline acetyltransferase showed no staining in the muscle layers or nerve fiber tracts of either part of the esophagus studied; however, selected neurons within the myenteric plexus of both regions (approximately 38%) were reactive. There was no significant difference in the number of positive choline acetyltransferase neurons in the lower esophageal sphincter or esophageal body.