DNA triplexes have been the subject of great interest due to their ability to interfere with gene expression. The inhibition of gene expression involves the design of stable triplexes under physiological conditions; therefore, it is important to have a clear understanding of the energetic contributions controlling their stability. We have used a combination of UV spectroscopy and differential scanning calorimetric (DSC) techniques to investigate the unfolding of intramolecular triplexes, d(A(n)C(5)T(n)C(5)T(n)), where n is 5-7, 9, and 11, and related triplexes with a single AT -> TA substitution in their duplex stem. Specifically, we obtain standard thermodynamic profiles for the unfolding of each triplex in buffer solutions containing 0.1 M or 1 M NaCl. The triplexes unfold in monophasic or biphasic transitions (triplex -> duplex -> coil) depending on the concentration of salt used and position of the substitution, and their transition temperatures are independent of strand concentration. The DSC curves of the unsubstituted triplexes yielded an unfolding heat of 13.9 kcal/mol for a TAT/TAT base-triplet stack and a heat capacity of 505 cal/degrees C center dot mol. The incorporation of a single substitution destabilizes triplex formation (association of the third strand) to a larger extent in 0.1 M NaCl, and the magnitude of the effects also depends on the position of the substitution. The combined results show that a single AT -> TA substitution in a homopurine/homopyrimidine duplex does not allow triplex formation of the neighboring five TAT base triplets, indicating that the in vivo formation of triplexes, such as H-DNA, is exclusive to homopurine/homopyrimidine sequences.