The sequence dependence of the double-stranded DNA (dsDNA)–binding affinity of TOTO, a thiazole orange dimer that functions as a DNA-intercalating fluorophore, was measured using single-molecule methods. An analysis was performed of the distribution of excited-state lifetimes of single molecules of TOTO intercalated into dsDNA fragments containing four–base pair sequences shown previously to have high affinity for TOTO under conditions used in nuclear magnetic resonance (NMR) spectroscopy. For the current studies, the putative binding sites were located centrally in 30–base pair–long dsDNA fragments in which the remaining sequence was either all poly-AT or poly-GC. The lifetime of TOTO fluorescence when bound to these fragments was entirely determined by the background sequence, i.e. DNA fragments with a poly-AT background predominantly gave a fluorescence lifetime of 1.7 ns, whereas DNA fragments with a poly-GC background gave a lifetime of 2.0 ns, independent of the presence or absence of the putative binding sequence. By performing competitive binding experiments in which these DNA fragments competed for TOTO binding with pure poly-AT fragments and using single-molecule fluorescence methods to determine the number of each type of DNA fragment having a TOTO bound in an equilibrium mixture, the relative binding affinity of each putative binding site was determined. The results of these experiments showed clearly that TOTO has no preference for binding to the putative binding sites over binding poly-AT or poly-GC under the conditions of these measurements. This suggests that there is very little sequence dependence of TOTO binding under conditions that would likely predominate in most biological applications of this intercalating dye.