N-Nitrose compounds, which are products of cellular metabolism, have been related to mutations and cancer. Here the influence of O⁴-methylthymine, a chemically modified base produced by the action of nitroso compounds has been studied. Long (1 ns) molecular dynamics simulations of four dodecanucleotides have been performed, two of which contain O⁴-methylthymine (base-paired with adenine in one and with guanine in the other), while the remaining two are the unmethylated versions of the former (one a ‘normal’ sequence, with T. A basepairs, and the other a sequence with G. T. mispairs). The simulation results are in good agreement with the available experimental data. A detailed structural analysis, which includes the hydrogen-bonding patterns, backbone torsion angles and basepair helical parameters indicates that the sequence containing methylthymine base-paired with guanine is more iso-structural with the normal sequence than the sequence in which methylthymine is base-paired with adenine. This may explain why guanine is incorporated opposite methylthymine upon replication. On the other hand, the methylthymine. adenine basepairs are unstable, leading to distortions in the helix. This should make the latter basepairs easier to recognize by the repair proteins than the mutated methylthymine.guanine basepairs.