The thymidine analogue DMAT was used for the first fluorescence-based study of direct, site-specific metal binding reactions involving unmodified nucleobases in duplex DNA. The fluorescence properties of DMAT-A base pairs were highly sensitive to mercury binding reactions at T-T mismatches located at an adjacent site or one base pair away. This allowed for precise determination of the local kinetic and thermodynamic parameters of T-HgII-T binding reactions. The on- and off-rates of HgII were surprisingly slow, with association rate constants (kon) ≈ 104–105 M–1 s–1, and dissociation rate constants (koff) ≈ 10–4–10–3 s–1; giving equilibrium dissociation constants (Kd) = 8–50 nM. In contrast, duplexes lacking a T-T mismatch exhibited local, nonspecific HgII binding affinities in the range of Kd = 0.2–2.0 μM, depending on the buffer conditions. The exceptionally high kinetic stabilities of T-HgII-T metallo-base pairs (half-lives = 0.3–1.3 h) perturbed dynamic processes including DNA strand displacement and primer extension by DNA polymerases that resulted in premature chain termination of DNA synthesis. In addition to providing the first detailed kinetic and thermodynamic parameters of site-specific T-HgII-T binding reactions in duplex DNA, these results demonstrate that T-HgII-T base pairs have a high potential to disrupt DNA metabolism in vivo.