Catecholaminergic cells are transiently present during development of the fetal murine bowel. These transient catecholaminergic (TC) cells appear at Day E10, but by Day E13 can no longer be detected. In order to evaluate the hypothesis that these cells are the precursors of enteric neurons, we investigated the possibilities that TC cells coexpress neuronal and catecholaminergic markers, that they can be found along the presumed path followed by crest-derived cells migrating to the gut, and that they are proliferating. TC cells were identified immunocytochemically using polyclonal or monoclonal antibodies to tyrosine hydroxylase (TH). At Day E9.5, TH-immunoreactive cells were observed to be present along the wall of the primordial esophagus in lines that extended from the developing nodose ganglia down to the boundary of the stomach. At Day E9.5, TC cells were absent from the remaining foregut. These lines of esophageal TH-immunoreactive cells became continuous with similar cells in the wall of the stomach and duodenum on Day E10. Coincident expression of neurofilament immunoreactivity was seen in all of the esophageal TH-immunoreactive cells present at Day E9.5, as well as in the entire set of esophageal and lower enteric TH-immunoreactive cells present at Day E10 (or later); moreover, at Days E9.5 and E10, all of the neurofilament-immunoreactive cells in the esophagus, stomach, or duodenum were also TH-immunoreactive. In contrast, neurofilament immunoreactivity was not expressed by the endodermally derived pancreatic duct and islet cells, which were also TH-immunoreactive; nor could expression of neurofilament immunoreactivity be detected in the TH-immunoreactive cells of the nodose ganglia. It was not until Day E11 that neurofilament-immunoreactive cells, which did not coexpress TH immunoreactivity (the definitive phenotype of enteric neurons) began to appear in the gut. Vagal axons reached as far distally as the nodose ganglion on Day E9.5, the esophagogastric junction on Day E10, and did not enter the stomach until Day E11. When the vagus nerves reached their level, the TH-immunoreactive cells in the wall of the esophagus came to lie among the nerve fibers. TH-immunoreactive cells are thus present on the pathway ultimately followed by the vagus nerves, but they develop before vagal fibers reach their level. The vagal TH-immunoreactive cells, therefore, are probably not initially migrating on vagal fibers, but appear instead to be overtaken by the descending vagus nerves. The TH-immunoreactive cells in the nodose ganglia and vagus nerves disappear in a proximodistal sequence and, except for vagal paraganglia, TH-immunoreactive cells are gone from the vagus nerves by Day E16. In addition to TH immunoreactivity, vagal and enteric TC cells were found to contain the immunoreactivity for dopamine β-hydroxylase (DBH) and aromatic l-amino acid decarboxylase. Unlike the transient TH-immunoreactive cells of the nodose ganglia, vagal and enteric TC cells displayed intense formaldehyde-induced histofluorescence, indicating that they contain an endogenous catecholamine. In addition, both types of TC cells accumulated [³H]norepinephrine ([³H]NE) by a mechanism that could be inhibited by desmethylimipramine. Vagal and enteric TC cells, like mature enteric neurons, also exhibited acetylcholinesterase activity and, in whole mount preparations, could be seen to have extended neurites that formed a network, apparently connecting clusters of the cells. Two hours after injection of bromodeoxyuridine, the label was detected immunocytochemically in the nuclei of a subset of both vagal and enteric TC cells …