Chromosomal deletions ('deficiencies') are powerful tools in the genetic analysis of complex genomes. They have been exploited extensively in Drosophila melanogaster, an organism in which deficiencies can be efficiently induced and selected. Spontaneous deletions in humans have facilitated the dissection of phenotypes in contiguous gene syndromes and led to the positional cloning of critical genes. In mice, deletion complexes created by whole animal irradiation experiments have enabled a systematic characterization of functional units along defined chromosomal regions. However, classical mutagenesis in mice is logistically impractical for generating deletion sets on a genome-wide scale. Here, we report a high- throughput method for generating radiation-induced deletion complexes at defined regions in the genome using ES cells. Dozens of deletions of up to several centiMorgans, encompassing a specific locus, can be created in a single experiment and transmitted through the germline. The ability to rapidly create deletion complexes along chromosomes will facilitate systematic functional analyses of the mammalian genome.