We examined the patterns of trefoil peptide gene expression in the ulcer-associated cell lineage (UACL) and mucosa adjacent to Crohn's disease in humans and during gastrointestinal adaptation to enteral feeding in rats. In the UACL, human spasmolytic polypeptide (hSP) mRNA and peptide are present in the acinar and proximal duct cells, whereas pS2 mRNA and peptide are found in the distal duct cells and in the surface cells. In mucosa adjacent to UACL, pS2 mRNA and peptide are expressed ectopically by goblet cells and neuroendocrine cells. Intestinal crypts associated with the UACL showed marked neuroendocrine cell hyperplasia. Ultrastructural immunolocalization showed pS2 to be copackaged in the mucous cell and neuroendocrine granules. The copackaging of a secretory protein in both mucous and neuroendocrine granules, which have different functions, is unusual and indicates an important role for pS2 in the secretory process itself or as a ligand delivered to its receptor via multiple routes. We also cloned the newest trefoil peptide, intestinal trefoil factor (ITF), from human and rat intestinal mucosa. Using in situ hybridization we demonstrated its synthesis by normal rat intestinal goblet cells. RNAse protection analysis revealed that the level of mRNA for rat ITF in small and large intestine was affected by the process of enteral feeding. We conclude that trefoil peptides are widely distributed in the intestine in human inflammatory bowel disease and are of considerable potential functional importance.