We investigated the thermodynamic stability of double-stranded DNAs with an oxidative DNA lesion, 2-hydroxyadenine (2-OH-Ade), in two different sequence contexts (5'-GA*C-3' and 5'-TA*A-3', A* represents 2-OH-Ade). When an A*-N pair (N, any nucleotide base) was located in the center of a duplex, the thermodynamic stabilities of the duplexes were similar for all the natural bases except A (N = T, C and G). On the other hand, for the duplexes with the A*-N pair at the end, which mimic the nucleotide incorporation step, the stabilities of the duplexes were dependent on their sequence. The order of stability is T > G > C >> A in the 5'-GA*C-3' sequences and T > A > C > G in the 5'-TA*A-3' sequences. Because T/G/C and T/A are nucleotides incorporated opposite to 2-OH-Ade in the 5'-GA*C-3' and 5'-TA*A-3' sequences, respectively, these results agree with the tendency of mutagenic misincorporation of the nucleotides opposite to 2-OH-Ade in vitro. Thus, the thermodynamic stability of the A*-N base pair may be an important factor for the mutation spectra of 2-OH-Ade.