Interstitial deletions of the chromosome 9p21 segment encoding the p16/CDKN2A tumor suppressor gene (i.e., 9p21 deletions) are frequently observed in a variety of human cancers. A majority of these deletions in lymphoid leukemia have been indicated to be mediated by illegitimate V(D)J recombination. In the present study, to elucidate the molecular processes of 9p21 deletions in nonlymphocytic malignancies, breakpoints for these deletions were analysed in 21 lung cancer cell lines and 32 nonlymphocytic cancer cell lines of nine other histological types. In all, 32 breakpoints in 21 lung cancer cell lines and 56 breakpoints in 32 nonlung cancer cell lines were mapped in a 450-kb segment encompassing the CDKN2A locus with a 10-kb resolution. The largest number of breakpoints (i.e., seven breakpoints in lung cancer and 12 breakpoints in nonlung cancers) was mapped in a 10-kb region containing the CDKN2A gene. More precise mapping of these seven and 12 breakpoints revealed that none of these breakpoints were located within 50-bp intervals to each other in this 10 kb region. Cloning and sequencing of breakpoints in 18 representative cell lines (six lung and 12 nonlung cancers) further revealed that there were no significant homologies among breakpoints in these 18 cell lines. In 11 (61%) cell lines, 1-5-bp nucleotides were overlapped at breakpoint junctions. These results indicate that DNA double-strand breaks triggering 9p21 deletions do not occur at specific DNA sequences, although they preferentially occur in or near the CDKN2A locus. It was also indicated that two broken DNA ends are rejoined by nonhomologous end-joining repair, preferentially utilizing microhomologies of DNA ends, in the occurrence of 9p21 deletions.