The distribution of fibronectin (FN), laminin (LM), Jl/tenascin, chondroitin sulphate proteoglycan (CSPG), neural cell adhesion molecule (NCAM), neurofilament (NF) and the HNK-1 epitope were studied immunohistochemically in the developing mid- and hindgut of E12.5-E16.5 rat embryos. Over this period the gut wall changed from a uniform mesenchyme to an annular organisation. FN and LM remained widely distributed, but Jl/tenascin became concentrated in mesenchyme outside the nascent circular muscle layer, and CSPG declined in, and NCAM increased in, the circular layer. Protease and fixation treatments suggested that CSPG could mask other molecules such as LM. This re-organisation proceeded bidirectionally, as a rostrocaudal wave which was met in the colon by a caudorostral wave. The caecum, however, was conspiciously delayed in all maturation events and also showed mesenchymal and serosal epithelial labelling for the cell-adhesion-related HNK-1 epitope, which was absent elsewhere. This period also covered the appearance of enteric neurons, recognised by HNK-1 and NF antibodies. Cells labelled by these antibodies appeared in a unidirectional rostrocaudal wave, from the duodenum at El2.5 to the rectum at El6.5. This wave was not in exact synchrony with the wave of intestinal maturation, but lagged behind so that neuronal cells first appeared in increasingly mature micro-environment at progressively more caudal levels. These cells initially were positioned imprecisely about mid-way across the gut mesenchyme layer and were not clearly related spatiotemporally to any of the above molecules. Slightly later, however, this neural region was broadly defined by relatively low levels of both CSPG and Jl/tenascin. The final position of the myenteric neurons was very precise, and was related to a thin Jl/tenascin layer and to a step in NCAM labelling intensity. Litters of pups, of which 25% would be expected to be spotting lethal homozygous embryos which develop total colonic and caecal aganglionosis, showed no difference in any of the molecules studied, but in 4 embryos out of 12, the progress of the rostrocaudal wave of neuron appearance was distinctly slowed even in the duodenum and proximal small intestine, regions well outside the final aganglionic zone. The observations suggest that this Hirschsprung’s-disease-like regionalised defect is a result of a generalised abnormality which does not involve gross changes in extracellular matrix and NCAM expression.