Trefoil peptides, a growing family of secretory molecules, have been identified mainly in the gastrointestinal tract of humans, rodents and amphibians. In the present study, the nucleotide sequence of a large portion (81%) of the gene encoding murine intestinal trefoil factor (mITF) and its whole genomic organization were determined. The mITF gene contains three exons distributed over 5 kb of genomic DNA. The genomic sequence is highly conserved, as compared with that of the rat and human ITF, and contains several AP-1-binding sites, the consensus binding site for the transcription factor Sp1, and a sequence homologous to a heat-shock element. Fluorescence in situ hybridization was used to assign ITF to chromosome 17 of the murine genome, a region syntenic with the trefoil gene cluster on human chromosome 21q22.3.