The mammalian Trefoil Factors (TFFs), TFF1/pS2, TFF2/SP and TFF3/ITF, are expressed and secreted throughout the gastrointestinal tract with a specific and complementary pattern. These proteins exhibit common functions in the protection and repair process of the gastrointestinal epithelial barrier. Here, we report the clustered organization of the three mouse TFF genes in a 40 kb DNA segment, in a head to tail orientation in the following order: TFF1, TFF2, and TFF3. Computer comparison of the mouse TFF promoter sequences to their human counterparts revealed conserved boxes in both mouse and human genes. Promoter methylation analyses showed that, in tissues where these genes are normally expressed, the proximal promoters of TFF1 and TFF2 are specifically not methylated and that of TFF3 is partially demethylated. In contrast, in organs that do not express TFFs, the promoters of the three genes are methylated. These findings strongly argue for the involvement of epigenetic mechanisms in the regulation of TFF expression in normal and pathological conditions.