BACKGROUND Most enteric neurones arise from neural crest cells that originate in the post-otic hindbrain, and migrate into and along the developing gastrointestinal tract. There is currently great interest in the possibility of cell therapy to replace diseased or absent enteric neurones in patients with enteric neuropathies, such as Hirschsprung's disease. However, it is unclear whether neural crest stem/progenitor cells will be able to colonize colon (i) in which the mesenchyme has differentiated into distinct layers, (ii) that already contains enteric neurones or (iii) that lacks a gene expressed by the gut mesenchyme, such as endothelin-3 (Et-3). METHODS Co-cultures were used to examine the ability of enteric neural crest-derived cells (ENCCs) from E11.5 mouse gut to colonize a variety of recipient hindguts. KEY RESULTS Enteric neural crest-derived cells migrated and gave rise to neurones in E14.5 and E16.5 aneural colon in which the external muscle layers had differentiated, but they did not migrate as far as in younger colon. There was no evidence of altered ENCC proliferation, cell death or neuronal differentiation in older recipient explants. Enteric neural crest-derived cells failed to enter most recipient E14.5 and E16.5 colon explants already containing enteric neurones, and the few that did showed very limited migration. Finally, ENCCs migrated a shorter distance and a higher proportion expressed the pan-neuronal marker, Hu, in recipient E11.5 Et-3(-/-) colon compared to wild-type recipient colon. CONCLUSIONS & INFERENCES Age and an absence of Et-3 from the recipient gut both significantly reduced but did not prevent ENCC migration, but the presence of neurones almost totally prevented ENCC migration.