Sequence-specific duplex DNA recognition by oligonucleotide-directed triple helix formation is a possible approach to in vivo gene inhibition. However, triple helix formation involving guanine-rich oligonucleotides is inhibited by physiological ions, particularly K+, most likely due to oligonucleotide aggregation via guanine quartets. Three oligodeoxynucleotide (ODN) derivatives were tested for their ability to resist guanine quartet-mediated aggregation, yet form stable triplexes. Electrophoretic mobility shift and dimethyl sulfate footprinting assays were used to analyze the formation of triplexes involving these oligonucleotide derivatives. In the absence of K+, all ODNs had similar binding affinities for the duplex target. Triplexes involving a 14mer ODN derivative containing 7-deazaxanthine substituted for three thymine bases or an 18mer ODN containing two additional thymines on both the 5' and 3' termini were abolished by 50 mM K+. Remarkably, triplexes involving an ODN derivative containing four 6-thioguanine bases substituted for guanine resisted K+ inhibition up to 200 mM. We hypothesize that the increased radius and decreased electronegativity of sulfur in the 6-position of guanine destabilize potential guanine quartets. These results improve the prospects for creating ODNS that might serve as specific and efficient gene repressors in vivo.