Background: The regional differences in the distribution of the trefoil peptides pS2 and human spasmolytic polypeptide (hSP) within the stomach are not known. The aim of this study was to gain insight into these functionally obscure molecules by characterizing their distribution. Methods: Tissue from gastrectomy specimens removed for peptic ulceration was examined to chart the distribution of hSP and pS2, using immuno-histochemistry and in situ hybridization to chart their messenger RNAs (mRNAs). Results: Colocalization of pS2 and hSP was noted in the gastric foveolar and surface epithelium throughout the stomach. In the gastric antrum and pylorus, an extremely strong hSP mRNA signal was present within pyloric-type glands; strong hSP immunostaining was also seen at this site and in mucous-neck cells. Neither pS2 mRNA nor pS2 peptide were shown within the deep portions of the pyloric glands. Within areas of intestinal metaplasia, a few goblet cells immunostained for pS2 and putative ulcer associated cell lineage was seen with a pattern of trefoil peptide localization similar to the ileum. Conclusion: The detailed function of these trefoil peptides is unknown, but their distribution suggests involvement in repair-enhancing mechanisms. hSP may be an important antral peptide and both of these peptides may play a specific reparative role.