Hirschsprung disease is the result of aganglionosis of a variable length of the terminal bowel, which arises from the incomplete colonisation of the embryonic gut by vagal neural crest-derived cells (NCC) that migrate caudally from the pharyngeal gut to the rectum. We have previously shown that a very small group of NCC, at the leading edge of this wave of migration, can proliferate and differentiate to innervate the entire distal gut. It remains unknown if this capability is unique to those cells at the leading edge of NCC migration. The hypothesis tested was that NCC capable of acting as stem cells are found throughout the developing enteric nervous system (ENS). Gut was taken from mice at embryonic day 11.5 as the leading edge of NCC migration enters the colon. Terminal colon was separated as aganglionic recipient gut and its rostral end juxtaposed to the caudal end of the small intestine or caecum. The explants were cultured on nitrocellulose filters for up to 120 h, after which time the apposed segments had fused. The gut was then fixed and examined by immunohistochemistry to detect the neuronal markers PGP9.5 and nitric-oxide synthase (NOS) to assess development of enteric ganglia. NCC migrated from the proximal gut into the terminal colon, colonising it along its entire length. The pattern of NCC colonisation and differentiation of NOS-positive neurons was the same, regardless of whether the NCC were derived from the leading edge of migration in the caecum or from more proximal regions of the small intestine. Vagal NCC have the capacity to migrate into separated aganglionic terminal colon and differentiate into neurons. NCC at the leading edge of migration and those located more proximally within the gut demonstrate equivalent ability to migrate to and differentiate in the terminal rectum. Further studies are required to confirm which of these migrating NCC have the properties of ENS stem cells.