Background: This work expands on recent observations that the trefoil peptides pS2 and human spasmolytic polypeptide (hSP) are expressed in the ulceration-associated cell lineage (UACL) glands developing in chronic ulcerative conditions. Methods: Trefoil peptide expression in small intestinal Crohn's disease was examined by in situ hybridization to reveal sites of expression of the messenger RNAs encoding pS2 and hSP and by immunohistochemistry and immunoelectron microscopy to localize the peptides in the UACL and adjacent goblet and neuroendocrine cells. Results: Goblet cells near the UACL expressed pS2 messenger RNA and peptide; ultrastructural immunolocalization revealed pS2 copackaged within mucous cell granules. Neuroendocrine cell hyperplasia was marked in crypts near the UACL; pS2 was copackaged with the neuroendocrine granules. Conclusions: Copackaging of a secretory protein, pS2, 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 receptors via different routes. It is concluded that trefoil peptides are of considerable potential functional importance in inflammatory bowel disease.