Reductively-activated mitomycin C (MC) presents a high specificity to the 5 -CG site and to a lesser extent the 5 -GG site. However, its affinity is different for each 5 -CG site. This was evidenced by using the 3 -5 exonuclease activity of T 4 DNA polymerase on a short DNA fragment exposed to MC, which was gradually activated by several Na 2 S 2 O 4 additions. The time-delayed appearance of some exonuclease digestion stop sites (corresponding to MC-monofunctional adducts) suggests that MC discriminates between very fine structural variations. The feature of the stop sites suggests a good fit of MC in the DNA groove, in the case of the major alkylation sites, but not in the case of a minor 5 -TG alkylation site. Furthermore, it is evidenced by the use of the chemical probe hydroxylamine (HA) that MC-monoalkylation of 5 -CG (or 5 -GG) does not induce notable local structural disturbance of the DNA double helix, as opposed to alkylation of the 5 -TG site of minor specificity, which leads to significant local DNA distortion. This suggests that the in vivo effect of MC is related, not only to amount of alkylated sites (essentially 5 -CG sites), but also to possible local DNA deformations (at minor alkylation sites).