Abstract We characterized a region of the mouse genome disrupted by integration of a gene trap (GT) vector in ES cells. On 5′ rapid amplification of cDNA ends analysis of the fusion transcripts containing the GT vector, we identified the eukaryotic protein synthesis initiation factor 4A1 gene ( Eif4a1) as a promoter-trapped gene. Plasmid rescue was used to show that the other end of the integrated vector disrupted the murine homolog of the human fragile X mental retardation syndrome-related protein 2 gene ( Fxr2h). Structural analysis of P1 clones, isolated from the wild-type mouse genome by PCR with Eif4a1-specific primers, indicated that the integration of the GT vector was accompanied by the deletion of about 35 kb of genomic DNA and that the disrupted region also included three genes, Cd68, Supl15h and Sox15, the latter two of which are transcribed in opposite directions with overlapping 3′ ends. These five different genes at least, Eif4a1, Cd68, Supl15h, Sox15 and Fxr2h, are clustered in a 40 kb region. The chromosomal location of this region was mapped by means of interspecific backcross panel DNAs to the central part of mouse chromosome 11, exhibiting a known region of synteny with human chromosome 17.