For understanding the mechanism of action of antitumor agents and designing new drugs, the DNA alkylating property of duocarmycin (DUM) and its analogues was examined. The thermal depurination products of calf thymus DNA covalently bonded to DUMA were revealed to be not only the DUMA-N3 adenine adduct but also unexpectedly the DUMA-N3 guanine adduct. In addition DUMSA and synthetic analogues, 1 and 2 with higher solvolytic stability, reacted more selectively with N3 adenine than DUMA did. The correlation between electrophilicity of the cyclopropane subunit in the molecule and selectivity to adenine was observed. KW-2189, the synthetic derivative of 1 which has improved in vivo antitumor activity, was designed as a prodrug requiring enzymatic hydrolysis of the carbamoyl moiety, followed by regeneration of 1. Surprisingly we discovered that KW-2189 itself alkylated DNA covalently without release of the carbamoyl moiety. For the mechanism of DNA alkylation by KW-2189, a novel alkylating reaction via the formation of an iminium intermediate 18 without loss of the carbamoyl moiety was proposed.