We have previously reported a method for making nested deletion complexes in mice by irradiation of ES cells. The key to this technology is that F1 hybrid ES cells (called v17.2) of the genotype (BALB/cTa × 129/SvJae) retain germline colonizing ability after exposure to levels of ionizing radiation that induce chromosomal deletions. In an effort to identify other genotypes of ES cells that are suitable for this technology, the radiation sensitivity of the cell line v6.4, which is of the genotype (C57BL/6J × 129/SvJae), was investigated. After treatment with a range of radiation exposures, the developmental potential of these cells was assayed by injecting them into blastocysts to generate chimeric mice. These experiments showed that while cell lethality increased as the level of radiation increased, the surviving ES cells retained full totipotency at all exposure levels, up to 400 Rads. Because polymorphism between parental microsatellite alleles in the F1 hybrid ES cells is important for ascertaining the sizes of induced deletions, the 129/SvJ and 129/SvJae allele sizes of 48 microsatellite loci on chromosome (Chr) 17 were determined. This revealed a higher level of polymorphism between 129 and C57BL/6J on Chr 17. The radiation tolerance, high polymorphism between parental strains, and presence of the widely used C57BL/6J strain component make v6.4 ES cells an attractive cell line for generating radiation-induced chromosomal deletions.