The hypothesis was tested that developing enteric neurons withdraw from the cell cycle in a sequence related to their phenotype. The birthdays of immunocytochemically identified myenteric and submucosal neurons were determined in the murine duodenum and jejunum. [3H]thymidine ([3H]TdR) was injected into timed pregnant mice or pups at 4-8 hour intervals over a 24 hour period. Pups were killed on postnatal day 30 (P30). [3H]TdR incorporation was detected by radioautography in enteric neurons, which were phenotypically identified by the simultaneous detection of the immunoreactivities of 5-hydroxytryptamine (5-HT), choline acetyl transferase (ChAT), neuropeptide Y (NPY), enkephalin (ENK), calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP). The dates of the earliest withdrawal from the cell cycle of neurons containing these markers were determined, as well as the length of time during which the identified neurons continued to be born, and the date on which their rate of birth was maximal. The birthdates of myenteric neurons that contained 5-HT (E8-E14, peak at E10) or ChAT (E8-E15, peak at E12) tended to be earlier than those that contained ENK (E10-E18, peak at E14), NPY (E10-E18, peak at E15), VIP (E10-P5, peak at E15), or CGRP (E10-P3, peak at E17). For any given immunocytochemically defined neuronal phenotype, submucosal neurons tended to be born later than their myenteric counterparts and submucosal neurons that contained neuropeptides were born later than those that contained only ChAT immunoreactivity. The day (E8) on which the first 5-HT- and ChAT-immunoreactive neurons became postmitotic is earlier than the day (E9) on which the colonization of the bowel by crest-derived cells has been detected. The population of neural precursors that colonizes the gut, therefore, is heterogeneous; many cells are proliferating, but a specific subset, which will ultimately give rise to serotoninergic or cholinergic neurons, is already postmitotic. Neurons continued to be born throughout fetal life and even after birth. Consequently, terminally differentiated neurons coexist in the developing enteric nervous system with dividing neural precursor cells. This observation is consistent with the idea that early developing neurons could affect the development of enteric neural precursors; moreover, they also demonstrate that it is possible to add neurons to the enteric plexuses even after the neural circuits on which the bowel depends have become functional.