We have assigned several mouse cDNA and genomic clones to the W region of mouse chromosome 5, established their position with respect to various marker loci in the region, and provided molecular verification that the W19H mutation is a deletion. Meiotic recombination analysis of an interspecific mouse backcross indicated the following gene order and distances [in centimorgans (cM)]: centromere-Emv-1-(13 cM)-D4S76-(17 cM)-D5SC25-(5 cM)-alpha-casein-(1 cM)-beta- casein-(6 cM)-alpha-fetoprotein-(18 cM)-beta-glucuronidase. D5SC25, an anonymous locus defined by a mouse brain cDNA, maps near the map position of W and within the breakpoints of the presumed genetic deletion that causes the W19H phenotype. Southern analysis of DNAs of W19H/+ interspecific F1 hybrid mice and somatic cell hybrid lines carrying the W19H deletion chromosome showed the deletion of D5SC25. In fact, analysis of other mutations at or near the W locus, which had been transferred from the strain of origin for many backcross generations, revealed the retention of donor restriction fragment length polymorphisms at the D5SC25 locus. Such evidence confirms close linkage between D5SC25 and W (within 1 cM) and indicates that the D5SC25 cDNA clone could serve as a starting point in a chromosome "walk" to W and other closely linked loci that affect development.