Benzopyridoindole and benzopyridoquinoxaline derivatives were conjugated to a 14-mer oligonucleotide at either of two different positions : the 5’ end or an internucleotide position in the center of the 14-mer. These oligonucleotide-intercalator conjugates were then tested for their ability to form stable DNA triple helices with a DNA target duplex under physiological conditions. All of the derivatives synthesized were found to do so. Two derivatives in particular, a benzo[h]pyridoquinoxaline (B[h]PQ) attached to the 5’ end and a benzo[e]pyridoindole (B[e]PI) attached to the internal position on the phosphate diester backbone, dramatically stabilized the triple helix under physiological conditions. In the absence of spermine, the melting temperature bf the tripler-to-duplex transition increased from 11 degrees C for a non-modified tripler to 38 and 37 degrees C, respectively, for the B [h]PQ and B[e]PI conjugates. Acridine-oligonucleolide conjugates were much less stable, melting at 25 degrees C (5’ attachment) and at 23 degrees C (internucleotide linkage). In the presence of spermine, the melting temperature increased from 28 degrees C for a nonmodified tripler to 51 and 54 degrees C for the B[h]PQ and B[e]PI conjugates, respectively, equivalent to a stabilization of similar to 4 kcal mol(-1) at 37 degrees C. Furthermore, the conjugation of these intercalators to the third strand was not detrimental to the selectivity of recognition of the target duplex sequence. Molecular modeling reinforced and provided possible models for some of the intercalator-triple helix interactions investigated. These results demonstrate the possibility for forming stable DNA triple helices at physiological pH and temperature.