A major gene for Hirschsprung's disease (HD) recently has been mapped in chromosome 10q11.2 and identified to be the RET proto-oncogene. Mutations of the RET gene have occurred in HD patients, and abnormalities of expression and function of Ret protein (a receptor tyrosine kinase, which is the product of the RET gene) have been found in their intestines. In vitro studies of the biological effects of HD mutations suggest a loss of function effect, which may be negative-dominant. However, the developmental role of the Ret protein in the organogenesis of the enteric nervous system (ENS) and its role in the pathogenesis of HD remain unclear. The authors present a study of the expression of Ret protein in the human ENS during fetal development. Fresh rectal tissues were obtained from nine fetuses (gestational age range, 12 to 22 weeks). Ret protein expression was studied immunohistochemically, using antibodies against the carboxy-terminal 20 amino acids (anti-Ret C) and the extracellular domain (anti-Ret R5). The tyrosine kinase activity of the fetal ENS was investigated with antiphosphotyrosine mouse monoclonal antibody against the phosphorylated tyrosine residues. Anti-Ret C immunostaining was observed in ganglion cells at all ages, but intense activity was significantly higher among the cells of the younger fetuses. Intense anti-Ret R5 immunostaining was present in the enteric ganglion cells of the 12-week-old fetus. The tyrosine kinase activity of ganglion cells increases progressively with advancing gestational age. The results of this study support the hypothesis that the Ret protein receptor might play a crucial role in the cellular and molecular processes involved in the development and maturation of the ENS, abnormalities of which could result in HD. High Ret protein expression and low tyrosine kinase activity have been reported to occur in small ganglia of the HD hypoganglionic segment. In the present study, these markers were typical of the primitive and immature ENS during the early phase of hindgut development.