We have developed a novel strategy, based on the random insertion by homologous recombination of artificial I-Sce I sites within mammalian repetitive DNA sequences, which should greatly facilitate the high resolution physical mapping of large DNA fragments cloned in YAC. A set of transgenic yeast strains containing appropriately spaced I-Sce I sites within the YAC insert defines a series of nested physical intervals against which new genes, clones or DNA fragments can be mapped by simple hybridisation. Sequential hybridisation using such a series of nested YAC fragments as probes can also allow the rapid sorting of phage or cosmid libraries into contigs. This approach, which has been applied to a YAC containing a 460 kb insert from the mouse X chromosome, may also have applications for the restriction mapping of large genomic segments, mapping of exons and the search for homologous genes.