The interactions between single- and double-stranded DNA and the trimethine cyanine homodimer dye, BOBO-3 (1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3 -dihydro-(benzo-1,3-thiazole)-2-methylidene]pyridinium tetraiodide), have been investigated in detail using absorption and steady-state and time-resolved fluorescence spectroscopy. The dye interacts with both single-stranded and double-stranded DNA, under a variety of conditions, with changes in its spectral characteristics. Our results indicated that the complex formed between BOBO-3 dye and DNA oligonucleotides could not be explained with a simple, single intercalation mechanism therefore, different modes of interaction were proposed. By using time-resolved fluorescence methodology and in-depth analysis of the fluorescence decay traces, we obtained the contribution of the different forms of BOBO-3: free in solution, a low affinity, electrostatically driven interaction with DNA, and a full bis-intercalation mechanism within the DNA double helix. With this information, we applied the McGhee-Von Hippel theory for two overlapping, noncooperative binding modes to obtain equilibrium binding constants and the number of sites Occupied for each binding mode. Binding constants for dye/dsDNA complexes in complete bis-intercalation and externally bound were (8.8 +/- 1.1) x 10(5) and (2.6 +/- 0.3) x 10(5) M-1, respectively. The corresponding recovered number of base pairs covered were 5.9 +/- 0.2 and 3.5 +/- 0.5 sites for each mode.