Reductive activation of the bifunctional alkylator and DNA cross-linking agent mitomycin C (MC) yields 2,7-diaminomitosene (2,7-DAM) as the major product of its activation both in cell-free systems and in vivo. 2,7-DAM lacks one of the alkylating functions of MC, the aziridine. We show that 2,7-DAM itself alkylates DNA monofunctionally upon reductive activation, to form a heat-labile adduct. A guanine-N7-2,7-DAM adduct was isolated from the drug-DNA complex upon heating. Nuclease digestion yielded this adduct both in the nucleoside (dG-2,7-DAM) and dinucleoside phosphate [d(GpG)-2,7-DAM] forms. The structures of all three forms were determined by LC-ESIMS and differential UV spectroscopy. The adduct is sequence-specific to guanines in (G)(n) tracts of DNA. A guanine-N7-2,7-DAM adduct in DNA was indirectly observed previously by Prakash et al. (Prakash, A. S.; Beall, H.; Ross, D.; Gibson, N. W. Biochemistry 1993, 32, 5518-5525). The results indicate that selective removal of the aziridine function of MC results in a switch from minor to major groove alkylation of DNA, and a switch of sequence specificity from guanines in CpG to guanines in (G)(n) tracts, They also show that the mitosene C-10 carbamate intrinsically reacts as an S(N)2 alkylating agent, specific to guanine-N7.