We constructed a yeast artificial chromosome (YAC) contig spanning the genes encoding Kit (Kit), the platelet-derived growth factor alpha receptor (Pdgfra), and fetal liver kinase 1 (Flk1), three members of a receptor tyrosine kinase gene family located in the central portion of mouse chromosome 5. The orientation of YAC clones and the extent of their overlap was determined by "probe content mapping," that is, hybridization analysis of YAC clones using the available gene probes and YAC end sequences. For four YAC clones, which constitute a minimal set spanning 1.8 Mb, a detailed restriction map was constructed. This map, in conjunction with the previously published long-range restriction map, indicates the order, the physical distances, and the relative transcriptional orientations of the Pdgfra, Kit, and Flk1 genes. The YAC clones and corresponding YAC end probes presented here provide an important resource for the molecular analysis of a cluster of developmental mutations, namely dominant white spotting (W), patch (Ph), recessive spotting (rs), and rump-white (Rw), associated with this chromosomal region.