Recently, it was reported that T-T mismatches can specifically recognize Hg(II), and form T-Hg(II)-T pairs. In order to understand the structure and properties of the T-Hg(II)-T pair, we measured NMR spectra for a DNA duplex, d(CGCGTTGTCC) x d(GGACTTCGCG), with two successive T-T mismatches (Hg(II)-binding sites) in the middle of the duplex. We identified imino proton resonances of the T-T mismatches in mercury-free duplex, and performed titration experiments with Hg(II) by using 1-dimensional (1D) (1)H NMR spectra. From the titration spectra, disappearances of imino proton signals were observed upon the addition of Hg(II). Furthermore, we observed additional signals of transient species, most likely mono-mercurated duplexes. This is an evidence that structural transformations between Hg(II)-free and Hg(II)-bound forms are slow enough for each species to give independent signals. These data strongly suggest that the imino protons of thymine bases were substituted with Hg(II), to form T-Hg(II)-T pairs in which one Hg(II) cross-links two N3 atoms of thymines.