DNA double-strand breaks (DSBs) are the most severe lesions induced by ionizing radiation, and unrejoined or misrejoined DSBs can lead to cell lethality, mutations and the initiation of tumorigenesis. We have investigated X-ray- and α-particle-induced mutations that inactivate the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in human bladder carcinoma cells and in hTERT-immortalized human fibroblasts. Fifty to 80% of the mutants analyzed exhibited partial or total deletions of the 9 exons of the HPRT locus. The remaining mutants retained unaltered PCR products of all 9 exons but often displayed a failure to amplify the HPRT cDNA. Hybridization analysis of a 2-Mbp NotI fragment spanning the HPRT gene with a probe 200 kbp distal to the HPRT locus indicated altered fragment sizes in most of the mutants with a wild-type PCR pattern. These mutants likely contain breakpoints for genomic rearrangements in the intronic sequences of the HPRT gene that allow the amplification of the exons but prevent HPRT cDNA amplification. Additionally, mutants exhibiting partial and total deletions of the HPRT exons also frequently displayed altered NotI fragments. Interestingly, all mutations were very rarely associated with interchromosomal exchanges analyzed by FISH. Collectively, our data suggest that intrachromosomal genomic rearrangements on the Mbp scale represent the prevailing type of radiation-induced HPRT mutations.