We examined the effect of mercury (II) cation on the thermal stability of heteroduplex and homoduplex. Addition of mercury (II) cation increased the melting temperature of heteroduplex containing T:T mismatch base pair by about 4 degrees C. The thermal stability of homoduplex and heteroduplexes containing other kinds of mismatch base pairs was not significantly changed by the addition of mercury (II) cation. Isothermal titration calorimetric study demonstrated that mercury (II) cation directly bound to T:T mismatch base pair in hcteroduplex at a molar ratio of 1:1. The binding constant and the enthalpy change for the binding of mercury (II) cation to T:T mismatch base pair was approximately 10(6) M(-1) and -6 kcal mol(-1), respectively. We conclude that mercury (II) cation directly binds to T:T mismatch base pair in heteroduplex with high affinity and specificity. Our results certainly support the idea that the addition of mercury (II) cation to T:T mismatch base pair in heteroduplex could be a convenient strategy for heteroduplex analysis and may eventually lead to progress in single nucleotide polymorphism genotyping.