Most poly(purine·pyrimidine) [poly(R·Y)] sequences in eukaryotic genomes are interrupted by one or more base pair inversions. When the inversions are centrally located, the poly(R·Y) sequences can be regarded as the sum of two abutting sites, each potentially capable of forming a triple helix. Employing band-shift, footprinting and modeling methods we examined the formation of triple helices at a critical 27 bp poly(R·Y) sequence interrupted by two adjacent CG inversions, and located in the promoter of the human bcr gene at transcription initiation. We designed several 13-mer and 14-mer triplex-forming oligonucleo-tides (TFOs) capable of binding the bcr abutting sites, thereby generating different base juxtapositions at the triple helical junction, to examine whether triplex formation occurs in a cooperative manner. It is found that in 50 mm Tris/HCl, pH 7.4, 10 mm MgCl2, 2 mM spermine, 37°C, the 13-and the 14-mer TFOs bind to one half of the bcr site with δG between -30 and -35 kJ·mol-1. However, when different 13-mer/14-mer combinations of TFOs were directed against the abutting poly(R·Y) sites, triplex formation has been found to be enhanced only for the triple helical junction formed by the 5′-A-T-3′ base juxtaposition, in keeping with a partial stacking suggested from modeling analysis. On the other hand, a longer 24-mer TFO, binding noncooperatively to the same abutting sites, forms a much more stable triplex (δG = -51 kJ·mol-1), notwithstanding the two T·CG triads in the middle. Modeling investigations reveal that there is no continuity or propagation of base stacking involving adjacent bases of the third strand at the site of base inversion as well as on the 5′ side. The data indicate that the entropy penalty of forming a triplex with two oligonucleotides is much higher than the energy gained from base stacking interactions at the triplex junction formed between the two TFOs.