Triple-helix recognition of the DNA major grove offers a powerful approach for the design of sequence specific DNA binding molecules. Although the triple-helix binding of pyrimidine containing third strands is well characterized now, the factors governing the recognition of double stranded DNA by GT containing third strands are poorly understood. Oligonucleotides with a variable amount of G and GpT or TpG steps have been synthesized here. A systematical study was performed to determine the influence of these changes on the triple-helix hybridization properties of these oligonucleotides. Large difference in the hybridization properties have been observed by band shift experiments.