Sequence-specific duplex DNA recognition by oligonucleotlde-dlrected triple helix formation Is a possible approach to In vivo gene inhibition. However, triple helix formation involving guanlne-rich oligo-nucleotides is Inhibited by physiological Ions, particularly K + , most likely due to oligonucleotide aggregation via guanine quartets. Three ollgodeoxynucleotlde (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 thymlnes on both the 5′ and 3′ termini were abolished by 50 mM K+. Remarkably, triplexes Involving an ODN derivative containing four 6-thloguanine 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-posltlon 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 .