The human FHIT gene is a putative tumor suppressor gene that maps to human chromosome band 3p14.2 in a region that is frequently deleted in cancers. It exhibits both genomic deletions and aberrant transcripts in a variety of tumors and spans the common fragile site FRA3B. This fragile site extends over a broad region of several hundred kb within the FHIT gene and may account for its instability in tumors. As one test of this hypothesis, we isolated the murine Fhit gene and asked whether it also contains a common fragile site and if it is unstable in mouse tumors or tumor cell lines. The Fhit gene was isolated, and the sequence was found to be 87.5% identical to that of the human FHIT gene in the open reading frame. Using fluorescence in situ hybridization, Fhit was assigned to mouse chromosome band 14A2, in a region that was previously shown to contain an aphidicolin-inducible mouse fragile site. Fluorescence in situ hybridization with genomic clones containing Fhit and flanking sequences demonstrated that gaps and breaks in the fragile site occur over a broad region within and proximal to the Fhit locus. Thus, the physical relationship of Fhit to a common fragile site is similar to that observed with the orthologous human FHIT gene and FRA3B.