The ability of non-ionic methoxyethylphosphoramidate (PNHME) α-oligodeoxynucleotides (ODNs), αdT₁₅ and αdCT dodecamer, to form triplexes with their double-stranded DNA targets was evaluated. Thermal stability of the formed complexes was studied by UV thermal denaturation and the data showed that these PNHME α-ODNs formed much more stable triplexes than phosphodiester (PO) β-ODNs did (ΔTm = + 20° C for αdCT PNHME). In addition, FTIR spectroscopy was used to determine the base pairing and the strand orientations of the triplexes formed by αdT₁₅ PNHME compared to phosphodiester ODNs with β or α anomeric configuration. While βdT₁₅ PO failed to form a triplex with a long βdA_n·βdT_n duplex, the Tm of the Hoogsteen part of the triplex formed by αdT₁₅ PNHME reached 40° C. Moreover αdT₁₅ PNHME displaced the βdT₁₅ strand of a shorter βdA₁₅·βdT₁₅ duplex. The αdCT PNHME and αdT₁₅ PNHME third strands were found antiparallel in contrast to αdT₁₅ PO which is parallel to the purine strand of their duplex target. The uniform preferential Hoogsteen pairing of the nucleotides αdT and αdC combining both replacements might contribute to the improve stability of the triplexes.