The striated muscle of the esophagus differs from other striated muscle, because it develops by the transdifferentiation of smooth muscle, and the motor end plates receive a dual innervation from vagal (cholinergic) motor neurons and nitric oxide synthase (NOS)-containing enteric neurons. Mash1−/− mice have no enteric neurons in their esophagus and die within 48 hours of birth without milk in their stomachs (Guillemot et al.[1993] Cell 75: 463–476). In this study, the innervation of the esophagus of newborn Mash1−/−, Mash1+/− and wild type mice was examined. There was no difference between Mash1−/−, Mash1+/−, and wild type mice in the transdifferentiation of the muscle and the development of nicotinic receptor clusters. However, there were significantly more cholinergic nerve terminals per motor end plate in Mash1−/− mice than Mash1+/− or wild type mice. Each of the Mash1−/− mice had fewer than 50 NOS neurons per esophagus, compared with approximately 3,000 in wild type mice. Newborn Mash1+/− mice also contained significantly fewer NOS neurons than wild type mice. In Mash1−/− mice, NOS nerve fibers were virtually absent from the external muscle but were present at the myenteric plexus. Unlike that of newborn wild type mice, the lower esophageal sphincter of Mash1−/− mice lacked NOS nerve fibers; this may explain the absence of milk in the stomach. We conclude that 1) the transdifferentiation of the esophageal muscle and the development of the extrinsic innervation do not require enteric neurons or MASH1, 2) extrinsic NOS neurons only innervate the myenteric plexus. J. Comp. Neurol. 408: 1–10, 1999.© 1999 Wiley-Liss, Inc.