The rump-white (Rw) mutation in the mouse was previously mapped as part of a cluster of spotting genes on Chromosome (Chr) 5 that includes the dominant spotting (W) and patch (Ph) loci. Recent studies have shown that the W locus encodes the KIT tyrosine kinase cell surface receptor and that Ph is a deletional mutation encompassing the platelet-derived growth factor receptor alpha subunit (Pdgfra) gene. However, the molecular basis of the Rw mutation remains to be established. We have analyzed an interspecific Mus spretus backcross segregating Rw and several loci proximal and distal to the W/Ph/Rw region to study the basis of this mutation. These studies indicated that loci within the En2 to Kit region of the chromosome do not recombine with one another even though they have been separated in other mapping studies presented here and elsewhere. We conducted a series of fluorescent in situ hybridization (FISH) studies with genomic probes to En2, Msx1, D5Buc1, and Kit to compare the physical order of these loci on the Rw and wild-type chromosomes. The Kit locus mapped to approximately the same region on both chromosomes of the Rw heterozygotes, while the positions of En2, Msx1, and D5Buc1 were reversed on the two chromosomes. Taken together, both the genetic and physical mapping data establish that the Rw mutation is associated with an inversion involving loci in the proximal region of Chromosome 5.