Binding reactions of HgII and AgI to pyrimidine-pyrimidine mismatches in duplex DNA were characterized using fluorescent nucleobase analogs, thermal denaturation and 1H NMR. Unlike AgI, HgII exhibited stoichiometric, site-specific binding of C–T mismatches. The on- and off-rates of HgII binding were approximately 10-fold faster to C–T mismatches (kon ≈ 105 M−1 s−1, koff ≈ 10−3 s−1) as compared to T–T mismatches (kon ≈ 104 M−1 s−1, koff ≈ 10−4 s−1), resulting in very similar equilibrium binding affinities for both types of ‘all natural’ metallo base pairs (Kd ≈ 10–150 nM). These results are in contrast to thermal denaturation analyses, where duplexes containing T–T mismatches exhibited much larger increases in thermal stability upon addition of HgII (ΔTm = 6–19°C), as compared to those containing C–T mismatches (ΔTm = 1–4°C). In addition to revealing the high thermodynamic and kinetic stabilities of C–HgII–T base pairs, our results demonstrate that fluorescent nucleobase analogs enable highly sensitive detection and characterization of metal-mediated base pairs – even in situations where metal binding has little or no impact on the thermal stability of the duplex.