The thermodynamic characterization of the stability of two DNA triple helices of alternate sequence was obtained by differential scanning calorimetry (DSC). The conformational properties of these triple helices were investigated by circular dichroism (CD) and molecular mechanics. The triplexes under investigation form by way of major groove Hoogsteen association of a Watson-Crick 16-mer duplex with an all pyrimidine 16-mer or 15-mer third strand. The target duplex is composed of two adjacent oligopurine-oligopyrimidine domains where oligopurine sequences alternate on the two duplex strands. Both the third strands contain a 3',3'-phosphodiester junction, which introduces the appropriate inversion of polarity and let the switch from one oligopurine strand of the duplex to the other. The two-third strands differ for the lack of a cytidine monophosphate in the junction region. Thermal denaturation profiles indicate the initial loss of the third strand followed by the dissociation of the target duplex with increasing temperature. Transition enthalpies, entropies and free energies were derived From DSC measurements. The comparison of Gibbs energies reveals that a more stable tripler is obtained when in the third strand there is the lack of one nucleotide in the junction region. The thermodynamic data were discussed in relation to structural models.