The gene coding for human intestinal trefoil factor (hITF), a recently described cellular motogen produced by gastrointestinal goblet cells and epithelia elsewhere, is a member of the rapidly growing trefoil peptide family. In a rodent-human somatic cell hybrid panel, the hITF (HGMW-approved symbol TFF3) genomic locus segregated with human chromosome 21q. Fluorescence in situ hybridization with a 2.1-kb genomic probe of the hITF gene mapped this locus more precisely to the q22.3 region. Triple fluorescence in situ hybridization, together with physical mapping of human genomic DNA using pulsed-field gel electrophoresis, revealed that the hITF gene is tightly linked to those encoding the other known human trefoil peptides, namely the breast cancer estrogen-inducable gene pS2 (BCEI) and human spasmolytic polypeptide (hSP/SML1). This gene family could become a useful marker for the genetic and physical mapping of chromosome 21 and for a better definition of the region involved in the clinical phenotype of several genetic diseases.