The unnatural enantiomer (ent) of the antitumor antibiotic mitomycin C (MC) forms both a mono- and a bisguanine adduct in DNA upon reductive activation, with the bisadduct constituting DNA interstrand cross-links. The structures of the two adducts were rigorously determined using less than 1 mg of available material. The adducts are diastereomers of the known major guanine adducts of natural MC : the 2 "-NH2 group is in the alpha- instead of the beta-configuration while the chirality of the 1 "-linkage to DNA guanine is cc, as with natural MC. The extent of DNA adduct formation was 20-50% of that of MC. Enzymatic reductive activation of both MC and ent-MC by NADPH-cytochrome c reductase proceeded at equal rates. Both the monoalkylation and the cross-linking reactions of ent-MC exhibited DNA-sequence selectivity for the CpG sequence in analogy to that by natural MC. This finding indicates that at this sequence activated MC and ent-MC both assume preferentially the "carbamate upstream" orientation along the DNA minor groove with respect to the target guanine, which exposes the prochiral C1 "-center of these drugs to a alpha-stereofacial aback by DNA guanine N-2. The results also reinforce the earlier proposal that prior to the covalent step a simple, common structural element, i.e., the 10 "-carbamate group, recognizes the CpG sequence by formation of a specific H-bond, and this enhances the rate of alkylation of the guanine at the CpG site. It is proposed that the fit of the 1 ",2 "-cis-substituted adducts resulting from ent-MC in the minor groove is not as favorable as that of the 1 ",2 "-trans-adducts of natural MC, and that this is the reason for the observed lower reactivity of ent-MC with DNA, as compared with MC. This lower reactivity to form DNA adducts may be the basis for the slightly (approximately 50%) lower cytotoxicity of ent-MC than of MC to tumor cells.