We have already found that a single silver (I) cation specifically binds to a single C:C mismatch base pair in heteroduplex, which increases the melting temperature (T(m)) of heteroduplex involving a single C:C mismatch base pair by about 4 degrees C. Here, to examine the properties of the interaction between two C:C mismatch base pairs and silver (I) cations, we analyzed the effect of silver (I) cations on the thermal stability of heteroduplexes involving two C:C mismatch base pairs. The positions of the two C:C mismatch base pairs in heteroduplex did not significantly affect the magnitude of the increase in T(m) upon addition of silver (I) cation, suggesting that the binding affinity of the first silver (I) cation with one of the two C:C mismatch base pairs and that of the second silver (I) cation with the other of the two mismatch base pairs may be independent of the positions of the two mismatch base pairs. The larger magnitude of the increase in T(m) for the second silver (I) cation binding than that observed for the first silver (I) cation binding suggests that the binding affinity for the second silver (I) cation may be significantly higher than that for the first silver (I) cation. Our results certainly support the idea that addition of the metal cation is a promising strategy for the mismatch base pair detection in the heteroduplex analysis and may eventually lead to progress in SNP genotyping.