Genes of the dilute-short ear (d-se) region of mouse chromosome 9 comprise an array of loci important to the normal development of the animal. Over 200 spontaneous, chemically induced and radiation-induced mutations at these loci have been identified, making it one of the most genetically well-characterized regions of the mouse. Molecular analysis of this region has recently become feasible by the identification of a dilute mutation that was induced by integration of an ecotropic murine leukemia virus genome. Several unique sequence cellular DNA probes flanking this provirus have now been identified and used to investigate the organization of wild-type chromosomes and chromosomes with radiation-induced d-se region mutations. As expected, several of these mutations are associated with deletions, and, in general, the molecular and genetic complementation maps of these mutants are concordant. Furthermore, a deletion breakpoint fusion fragment has been identified and has been used to orient the physical map of the d-se region with respect to the genetic complementation map. These experiments provide important initial steps for analyzing this developmentally important region at the molecular level, as well as for studying in detail how a diverse group of mutagens acts on the mammalian germline.