Retrograde and anterograde tracing and immunohistochemical techniques were used to examine the origin of the extrinsic innervation, and the development of the vagal innervation to the mouse esophagus. Cholinergic nerve terminals were localised using an antiserum to the vesicular acetylcholine transporter and cholinergic cell bodies were localised using an antiserum to choline acetyltransferase. Cholinergic nerve terminals, which also contained calcitonin gene-related peptide, were present at the motor end plates in the external (striated) muscle of the esophagus. Following injection of Fast Blue into subdiaphragmatic or cervical levels of the esophagus, the only retrogradely-labelled cholinergic nerve cell bodies that also contained calcitonin gene-related peptide were found in the nucleus ambiguus. Neurons in the dorsal motor nucleus of the vagus, the nodose ganglia and dorsal root ganglia gave rise to a number of different types of nerve terminals within the myenteric plexus. Retrogradely-labelled neurons in the dorsal motor nucleus of vagus contained cholinergic markers only, nitric oxide synthase only or cholinergic markers plus nitric oxide synthase, retrogradely-labelled neurons in the dorsal root ganglia contained calcitonin gene-related peptide only, and a small number of retrogradely-labelled neurons in the nodose ganglia contained tyrosine hydroxylase. The development of the vagal innervation to the esophagus was examined following application of DiI to the vagus nerve of fixed mouse embryos. Anterogradely-labelled nerve fibres, which arose from both nodose ganglia and the medulla, were already present in the esophagus of embryonic day 12 (E12) mice. Some of the DiI-labelled vagal nerve fibres were present in among the smooth muscle cells of the external muscle layer prior to their transdifferentiation to striated muscle. We conclude that the neurons in the nucleus ambiguus that project to the esophagus differ from other extrinsic neurons in their chemistry as well as their targets within the esophagus. The development of the extrinsic innervation precedes the transdifferentiation of the external muscle to striated muscle, raising the possibility that, during development, smooth muscle of the esophagus is innervated transiently by vagal neurons.