Glial cell line-derived neurotrophic factor (GDNF) is a ligand for the receptor complex of GDNF family receptor alphas (GFRalphas) and Ret receptor tyrosine kinase, the product of a known Hirschsprung's disease gene. The aim of this study was to analyze the mRNA distribution of these genes in the developing human intestine to understand their roles in enteric innervation.Cryosections of fetal and newborn stomach, ileum, and colon were hybridized in situ with S35-labeled cRNA probes to GDNF, Ret, GFRalpha-1 or GFRalpha-2. GDNF mRNA levels in fetal ileum and colon were compared by reverse transcription-polymerase chain reaction (PCR).GDNF mRNA expression was abundant in the muscularis mucosae of both fetal and newborn colon but was found neither in the neural plexuses nor in other regions of the intestine. Accordingly, by reverse transcription-PCR, GDNF mRNA level was many times higher in colon than ileum. Ret, GFRalpha-1 and GFRalpha-2 mRNA were expressed in the ganglionic cells of both myenteric and submucosal plexuses throughout the intestine.The highly restricted distribution of GNDF mRNA suggests an important role for muscularis mucosae in the development of human enteric nervous system. Ret, GFRalpha-1, and GFRalpha-2 most likely act as GDNF receptors in colon but may have alternative ligands in other enteric segments.