Many early investigations on triple helices have been devoted to the study of the triplex formed by dT*dA-dT base triplets in which the third strand is oriented parallel to the dA strand. We now describe an intramolecular triple helix with dT*dA-dT base triplets in which the pyrimidine third strand is oriented antiparallel, formed by folding back twice the tridecamer dT10-linker-dA10-linker-dT10 (linker = pO(CH2CH2O)3p). Third-strand base pairing to the target strand, sugar conformation, and thermal denaturation of the triplex have been studied by Fourier transform infrared spectroscopy. Our results confirm than when the third-strand orientation is reversed from parallel to antiparallel with respect to the target strand, the third-strand hydrogen-bonding scheme is changed from Hoogsteen to reverse Hoogsteen. The sugar conformation in this triple helix is of the S type (C2'endo/anti, B family form) from all strands. Our results are discussed with respect to models for triplexes proposed as intermediates in homologous recombination [Zhurkin, V.B., Raghunathan, G., Ulyanov, N.B., Camerini-Otero, R.D., & Jernigan, R.L. (1994) J. Mol. Biol. 239, 181-200].