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A method of generating mice from embryonic stem (ES) cells with a large chromosomal deletion produced by X-ray irradiation has been developed. Fifty-two mutant ES clones were made that carried a nested set of chromosomal deletions up to approximately 10 cM in length around the hprt locus on the X Chromosome (Chr). Germline chimeras were generated from three ES clones with deletions ranging from 200 to 700 kb. In germline male mice from two independent clones, deletions around the hprt locus yielded a runty phenotype or caused death at birth. The runty mice had approximately 1/3 the body weight and size of wild littermates and did not survive more than 3 weeks after birth. The most plausible cause of these phenotypes is defects in regions flanking the hprt locus. This method of creating mutant mice with a large chromosomal deletion is very useful for the identification and understanding of gene functions.