The thiazole orange derivative, TO-PRO-3, and its dimeric analogue, TOTO-3, bind strongly to both double- and single-stranded DNA. Both the bound and the free dye undergo efficient nonphotochemical hole burning. From the structure of the hole burned and the fluorescence line-narrowed spectra, it is concluded that there are multiple modes of binding of the dyes to DNA. burned vibronic band structure are similar to those of the free dye. It is proposed that the molecules in this mode are externally bound to the DNA chain. Molecules in the other binding mode are characterized by a strong interaction with DNA. These are proposed to be molecules in intercalated or base-stacked configurations.