We have used one and two dimensional 1H NMR spectroscopy to characterize the binding of a homodimeric oxazole yellow dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4-( 3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene)-quinoliniu m tetraiodide (YOYO), to oligonucleotides containing the (5'-CTAG-3')2 and the (5'-CCGG-3')2 binding sites in either different oligonucleotides or in the same oligonucleotide. YOYO bis-intercalates strongly in all the oligonucleotides used and binds preferentially to a (5'-CTAG-3')2 binding site in the oligonucleotide d(CGCTAGCG)2 (1). YOYO also binds preferentially to a (5'-CCGG-3')2 sequence in the oligonucleotide d(CGCCGGCG)2 (2) but slightly less favorably than to the (5'- CTAG-3')2 sequence in 1. The binding of YOYO to the d(CGCTAGCCGGCG):d(CGCCGGCTAGCG) (3) oligonucleotide, containing two preferential binding sites, was also examined. YOYO forms mixtures of 1:1 and 1:2 complexes with oligonucleotide 3 in ratios dependent on the relative amount of YOYO and the oligonucleotides in the sample. The binding of YOYO to the oligonucleotide 3 occur sequence selective in the (5'-CTAG-3')2 site and the (5'- CCGG-3')2 site. We have also used two dimensional 1H NMR spectroscopy to determine the solution structure of the DNA oligonucleotide d(5'-CGCTAGCG-3')2 complexed with YOYO. The determination of the structure was based on a total relaxation matrix analysis of the NOESY cross peaks intensities. DQF-COSY spectra were used to obtain coupling constants for the deoxyribose ring protons. The coupling constants were transformed into angle estimates. The NOE derived distance and dihedral restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the YOYO-complex from both A-form and B-form dsDNA starting structures giving a total of 40 final structures. Since many NOE contacts were observed between YOYO and dsDNA the resulting structure has a fairly high resolution and allows determination of local features in the dsDNA structure after YOYO binding. The root-mean-square (rms) deviation of the coordinates for the forty structures of the complex was 0.39 A. The local DNA structure is distorted in the complex. The helix is unwound by 106 degrees and has an overall helical repeat of 13 base pairs caused by the bis-intercalation of YOYO. The polypropylene amine linker chain is located in the minor groove of dsDNA. Even though the YOYO chromophore contains an oxygen atom instead of the larger sulphur atom in the corresponding compound, TOTO, the structures establish that YOYO require more space than TOTO in the intercalation sites. This is probably caused by the more rigid and planar chromophores in YOYO compared to TOTO.