We quantitatively analyzed the thermodynamic contributions of 12-base pairing to DNA duplex dissociation and association using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). DNA duplexes consisting solely of A-T base pairs were systematically replaced with G-C base pairs and their thermodynamic properties were analyzed. Circular dichroism confirmed the formation of B-DNA, and DSC revealed that both the melting temperature and calorimetric enthalpy change increased in proportion to GC content, indicating enhanced thermal stability, mainly because of base stacking interactions. The more stable structure of DNA with high GC content was also supported by oligonucleotide binding analysis using ITC. The ITC titration profiles showed that the binding enthalpy of the oligonucleotide to its complementary strand initially reached a maximum exothermic value and then stabilized, suggesting a transition from a flexible single-stranded conformation to a more ordered duplex structure.