Triplex-forming oligonucleotides are able to modulate gene expression by site-specific binding to genomic DNA. Their use as therapeutic agents is limited by inefficient cellular uptake, scarce nuclear internalization, and oligonucleotide self-aggregation. In this study, we demonstrate that a 13-mer AG motif oligonucleotide covalently linked to a high-molecular mass (9000 Da) polyethylene glycol (PEG ODN₁₃) exhibits uptake and biological properties that are superior to those of the nonconjugated isosequence analogue (free ODN₁₃). Band-shift and footprinting experiments showed that PEG ODN₁₃ forms a stable triple helix (apparent K_d between 10^-6 and 10^-7 M in 50 mM Tris-acetate, 10 mM MgCl₂, pH 7.4, 37 °C) with a natural polypurine-polypyrimidine target located in the 5‘ flanking region of the human bcr/abl oncogene. Confocal laser microscopy performed on unfixed live cells stained with hexidium iodide as well as on glass-fixed cells stained with propidium iodide showed that fluorescein-labeled PEG ODN₁₃ is far more efficiently taken up and internalized in the nucleus by K562 and HeLa cells than the nonconjugated free ODN₁₃. It was found that PEG ODN₁₃ specifically downregulated the transcription of bcr/abl mRNA at 65 ± 5% with respect to control and inhibited cell growth by 32 ± 3% in a 3 day liquid culture assay. Moreover, PEG ODN₁₃ was more resistant against S1 and fetal bovine serum nucleases than free ODN₁₃, and less inclined to self-associate into multistrand structures in solution. Taken together, these results provide useful elements for designing artificial transcription repressors with enhanced potency in vivo.