Trefoil peptides are small secretory proteins characterized by three intrachain disulfide bonds forming the trefoil motif or P-domain. They are abundantly expressed on mucosal surfaces, especially of the gastrointestinal tract. In pathological conditions such as ulcers, metaplasia and neoplasia, their expression is upregulated. Three human trefoil peptides have been described: the estrogen-inducible pS2 protein, the spasmolytic protein and the intestinal trefoil factor. Recently, their role in the maintenance of surface integrity and ulcer healing was discussed. We already mapped the corresponding three genes (BCEI), SML1, TFF3) to the same genomic region (21q22.3). In this paper, we show that the three genes are clustered in a tandemly orientated fashion within 50 kb on a bacterial artificial chromosome (BAC) recombinant. This cluster is located adjacent to D21S19 and the locus order is cen-D21S212-TFF3-SML1-BCEI-D21S19-tel, whereas transcription of all three genes is directed towards the centromere. The gene structure of SML1 exhibits four exons, two of which encode the two separate trefoil motifs. TFF3 and BCEI, both containing one trefoil motif, are composed of three exons each, suggesting gene duplication and exon-shuffling events during evolution. The 5'-flanking region of SML1 was compared to the corresponding region of other trefoil genes. Two motifs with identical sequence and positions are shared between SML1 and BCEI, thus presenting possible targets for stomach-specific gene regulation. Two other motifs are shared within all known human and rat trefoil genes, suggesting a coordinated regulation and/or a common locus-controlling region. Using RT-PCR, a change in the pattern of trefoil gene expression is detected in tissue samples from normal gastric mucosa, hyperplastic polyps, gastric cancer, and gastric cancer cell lines, respectively.