Human enteric innervation was studied immunochemically with neuron-specific-enolase (NSE), a specific neurone marker indicative of differentiation, and substance P, a potent member of the family of neuropeptides. By examining various levels of the gut in 28 normal human fetuses of gestational ages 9 to 21 weeks, we showed that enteric neurones as a whole, as well as peptidergic neurones in particular, followed a dual gradient of development proceeding from each end to the middle of the gut. This suggests the need for caution in accepting the hypothesis of the pathogenesis of Hirschsprung's disease based on the concept of a single craniocaudal gradient of enteric neuronal development. In studies of six infants with Hirschsprung's disease, NSE immunostaining was found to be potentially useful for diagnostic purposes. NSE activity suggested that the hypertrophied nerve bundles in aganglionic bowel were metabolically active and functionally mature. Substance P-immunoreactivity was decreased in both aganglionic and distal ganglionic bowel in Hirschsprung's disease, suggesting that substance P-nerves were more extensively affected developmentally than other enteric neurones. In 28 infants with pyloric stenosis (IHPS), the presence of intense NSE activity in the ganglia in the pylorus suggested that these neurones were neither immature nor severely degenerated. A decrease in substance P immunoreactivity in IHPS suggested possible involvement of peptidergic innervation in the pathogenesis of IHPS.