The 643 transgenic mouse line carries an autosomal dominant insertional mutation that results in hemifacial microsomia (HFM), including microtia and/or abnormal biting. In this paper, we characterize the transgene integration site in transgenic mice and preintegration site of wildtype mice. The locus, designated Hfm (hemifacial microsomia-associated locus), was mapped to chromosome 10, B1-3, by chromosome in situ hybridization. We cloned the transgene insertion site from the transgenic DNA library. By using the 5' and 3' flanking sequences, the preintegration region was isolated. The analysis of these regions showed that a deletion of at least 23 kb DNA occurred in association with the transgene integration. Evolutionarily conserved regions were detected within and beside the deleted region. The result of mating between hemizygotes suggests that the phenotype of the homozygote is lethality in the prenatal period. These results suggest that the Hfm locus is necessary for prenatal development and that this strain is a useful animal model for investigating the genetic predisposition to HFM in humans.