Streptozotocin (STZ) is known to induce insulin-dependent diabetes mellitus via DNA damage in experimental animals. The mechanism of induction of DNA damage by STZ was investigated in vitro, using a human cell line and 32P-labeled DNA fragments isolated from human genes. STZ induced cellular DNA damage and apoptosis, and frequently initiated DNA modification at guanines, especially at the middle guanine in runs of three and at the guanine at the 3'-end of runs of two guanines, similar to N-methyl-N-nitrosourea, a typical methylating agent. Scavengers for reactive oxygen species or nitric oxide did not inhibit the induction of DNA damage by STZ. On the other hand, damage induction was inhibited by sodium acetate and sodium chloride, which can reduce the reactivity of methylating agents to DNA via the sodium cation. These results suggest that STZ induces DNA damage by methylation of guanines via methyl cations. This alkylation may be responsible for triggering apoptosis, and subsequently diabetes.