A restriction enzyme, MspI, was introduced into cultured human cells as a probe to detect genomic regions susceptible to DNA double-strand breaks (DSBs). A 2 h exposure to MspI at a concentration of 8 U/mul produced DSBs at MspI sites in more than 80% of HeLa cells. The sensitivity to digestion was examined on chromosomal DNAs for the region containing the p16 tumor suppressor gene and two other related genes, p14ARF and p15, by Southern blot hybridization analysis and linker-mediated capture of DNA fragments digested in vivo. DNAs for the promoter regions of the three genes, respectively, were sensitive to MspI digestion in HeLa cells, while DNA for the p16 promoter region was less sensitive in lung cancer cells with hypermethylation of the region. Breakpoints for interstitial 9p21 deletions removing the p16/p14ARF/p15 locus in a variety of human cancers were significantly over-represented in the three sensitive regions. The results suggest that the MspI sensitivity in vivo of each genomic region reflects its susceptibility to DSBs that trigger chromosome aberrations in human cells. This method could help us understand the pathogenic significance of differential susceptibility to DSBs among genomic regions in human carcinogenesis.