We report the first example of a stable triple-stranded helix consisting of exclusively T . AT (reverse-Hoogsteen . Watson-Crick) base triplets. The orientation of the third (T) strand in this tripler is anti-parallel with respect to the purine strand of the underlying duplex. Previous studies have examined the formation of these "antiparallel" T . AT triplets within a G . GC-rich environment; however, the present study demonstrates that G . GC triplets are not a requirement. Our approach to induce and stabilize the antiparallel tripler involves the use of a branched oligonucleotide with two parallel dT(10) strands joined to riboadenosine via 2’-5’ and 3’-5’ phosphodiester Linkages, i.e., rA([2’-5’-dT10])3’-5’-dT(10) (1) (Hudson, R. H. E.; Damha, M. J. J. Am. Chem. Sec. 1993, 115, 2119-2124). This tripler was further stabilized by MgCl2 or NaCl at neutral pH. Triple helix formation by branched oligonucleotide 1 and dA(10) was investigated by thermal denaturation analysis and circular dichroism spectroscopy. The melting curves at 260 and 284 nm show a single transition from bound to unbound species, indicative of cooperative melting. A linear oligonucleotide with a loop made of four dC residues between two dT(10) strands, and with a 5’-5’-phosphodiester linkage at one of the C/T-10 junctions, i.e., 3’-dT(10)C(4)-5’-5’-dT(10)-3’, did not form a similar triple helical structure. This results shows that the conformational rigidity imparted to the pyrimidine strands, by the branch point in 1, serves to preorganize and stabilize the complex. Potsssium ions inhibited triplex helix formation. In accord with what has been demonstrated previously for " parallel" Py . PuPy (Hoogsteen-Watson-Crick) triplexes, we show that short oligoadenylates (i.e., dA(4) and dA(5)) can bind cooperatively to the branched oligomer 1. The triplex-inducing capacity of branched oligonucleotides has potentially important implications in the study of intramolecular triplexes that occure in vivo.