Background and Aims: The lower esophageal sphincter is innervated primarily by enteric motor neurons. The somata of excitatory and inhibitory motor neurons were identified and mapped. Methods: Retrograde labeling in organotypic culture and immunohistochemistry were used to identify motor neuron somata. Results: 1,1'-Didodecyl-3,3,3',3'-tetramethyl iedocarbocyanine perchlorate (Dil) on the left side of the sphincter labeled descending motor neurons located up to 26 mm along the esophagus and locally (within 2 mm) and gastric motor neurons. Dil applied to the right side of the sphincter labeled descending and local motor neurons but very few gastric motor neurons. Inhibitory motor neuron cell bodies, identified by nitric oxide synthase immunoreactivity, accounted for 86% ± 4% (n = 6) of descending motor neurons and 53% ± 4% of local motor neurons labeled from the right side. Excitatory motor neurons, immunoreactive for choline acetyltransferase, accounted for 20% ± 3% (n = 6) of descending motor neurons and for 47% ± 4% of local motor neurons. All motor neurons were unipolar, but inhibitory motor neurons were significantly larger than excitatory neurons. Conclusions: The lower esophageal sphincter is innervated by local excitatory and inhibitory motor neurons and by descending esophageal inhibitory neurons. The oblique muscle, supplied by gastric motor neurons, is closely associated with the gastroesophageal junction.