The application of fluorescence in situ hybridization (FISH) using whole-chromosome paints (WCPs) is proving to be a very powerful technique for revealing chromosomal instability that, for the most part, has gone undetected by conventional cytogenetic analysis. We have analyzed the frequency of translocations in lymphocytes and lymphoblastoid cell lines from ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) homozygotes and heterozygotes using a three-color chromosome-painting technique (WCP 1, 2, 4). With this assay we were able to detect an increased frequency of spontaneous translocations in AT homozygotes (median, 18.47 ± 10.82 translocations per 1,000 metaphase cells; 10 patients) and AT heterozygotes (median, 7.87 ± 3.15 translocations per 1,000 cells; 7 patients), in comparison to controls (median, 2.26 ± 1.75 translocations per 1,000 cells; 10 controls). Analysis of NBS homozygotes (median, 19.05 ± 11.27 translocations per 1,000 cells; 5 patients) and NBS heterozygotes (median, 6.93 ± 3.04 translocations per 1,000 cells; 6 patients) also showed an increased frequency of translocations in these patients compared to controls. The presence of such hitherto undetected chromosomal aberrations corroborate previous findings of spontaneous chromosomal instability in AT and NBS patients, as manifested by an increased rate of open breaks and rearrangements involving chromosomes 7 and 14. Moreover, we show that the degree of genomic instability in AT and NBS patients is even higher than previously established and that some AT and NBS heterozygotes evidence spontaneous chromosomal instability as well. These increased levels of nonspecific translocations could be an important risk factor for the development of malignancies in homozygotes and heterozygotes for ATM or NBS1 gene mutations.