The terminal portion of the bowel of the lethal spotted mutant mouse (ls/ls) lacks an enteric nervous system due to the failure of neural crest precursors to colonize this region during embryonic life. As a result, the mouse develops congenital megacolon. We have postulated that the defect occurs because the microenvironment of the aganglionic segment is segmentally abnormal and does not permit the migration and/or survival of the enteric neural or glial precursors in the affected zone. We have examined the terminal segment of adult ls/ls and control mice by light and electron microscopy to determine if the defect is associated with identifiable structural abnormalities that persist to maturity. A striking abnormality is an overgrowth of the muscularis mucosa in the adult ls/ls mouse, particularly in the outer longitudinal layer. Electron microscopy also reveals an extensive thickening of the basal lamina around smooth muscle cells. In addition, nerves that are derived from fibers that are extrinsic to this area are abnormal. Large bundles of nerve fibers, some of which contain myelinated axons, large-caliber unmyelinated axons, and abundant collagen, are prominent in the intermuscular region of the aganglionic segments and often reach into the submucosa. The supporting cells of the unmyelinated and myelinated nerves in the aganglionic segment have voluminous perineural cytoplasm typical of immature Schwann cells. They also exhibit intermediate filaments in their cytoplasm. Otherwise they have the typical morphology of peripheral Schwann cells, rather than enteric glia, including individual ensheathment of axons and a surrounding basal lamina. We suggest that the extracellular matrix and/or cells of mesenchymal origin of the terminal bowel of the ls/ls mouse may prevent the ingrowth of the normal precursors of the glia as well as neurons of the enteric nervous system, but may permit or even encourage the ingrowth of abnormal numbers of extrinsic axons.