The lethal spotted (ls) mouse has been used as a model for the human disorder Hirschsprung’s disease, because as in the latter condition, ls/ls homozygotes are born without ganglion cells in their terminal colons and, without surgical intervention, die early as a consequence of intestinal obstruction. Previous studies have led to the conclusion that hereditary aganglionosis in ls/ls mice occurs because neural crest-derived enteric neuroblasts fail to colonize the distal large intestine during embryogenesis, perhaps due to a primary defect in non-neuroblastic mesenchyme rather than migrating neuroblasts themselves. In this investigation, the latter issue was addressed directly, in vivo, by comparing the distributions of ls/ls and wild-type neurons in aggregation chimeras. Expression of a transgene, D H-nlacZ, in enteric neurons derived from the vagal neural crest, was used as a marker for ls/ls enteric neurons in chimeric mice. In these animals, when greater than 20% of the cells were wild-type, the ls/ls phenotype was rescued; such mice were neither spotted nor aganglionic. In addition, these ‘rescued’ mice had mixtures of ls/ls and wildtype neurons throughout their gastrointestinal systems including distal rectum. In contrast, mice with smaller relative numbers of wild-type cells exhibited the classic ls/ls phenotype. The aganglionic terminal bowel of the latter mice contained neither ls/ls nor wild-type neurons. These results confirm that the primary defect in ls/ls embryos is not autonomous to enteric neuroblasts, but instead exists in the non-neuroblastic mesenchyme of the large intestine.