Double-stranded DNA offers multiple binding sites to DNA stains. Measurements of noncovalently bound dye-nucleic acid complexes are, necessarily, measurements of an ensemble of chromophores. Thus, it is difficult to assign fluorescence properties to base-pair-specific binding modes of cyanine dyes or, vice versa, to obtain information about the local environment of cyanines in nucleic acids by using optical spectroscopy. The feasibility to stain DNA and simultaneously probe local perturbations by optical spectroscopy would be a valuable asset to nucleic acid research. So-called FIT probes (forced intercalation probes) were used to pinpoint the location of the DNA stain thiazole orange (TO) in PNADNA duplexes. A detailed analysis of the base-pair dependence of optical properties is provided and enforced binding of TO is compared with "classical" binding of free TO-PRO1. UV-visible absorbance, circular dichroism (CD) and fluorescence spectroscopy, and melting-curve analyses confirmed site-specific TO intercalation. Thiazole orange exhibited base-specific responses that are not observed in noncovalent dye-nucleic acid complexes, such as an extraordinary dependence of the TO extinction coefficient (+/-60 % variation of the averaged epsilon(max) of 57,000 M(-1) cm(-1)) on nearest-neighbor base pairs. TO signals hybridization, as shown by increases in the steady-state fluorescence emission. Studies of TO fluorescence lifetimes in FIT-PNA and in DNADNA and PNADNA complexes highlighted four different fluorescence-decay processes that may be closed or opened in response to matched or single-mismatched hybridization. A very fast decay process (0.04-0.07 ns) and a slow decay process (2.33-3.95 ns) provide reliable monitors of hybridization, and the opening of a fast decay channel (0.22-0.48 ns) that resulted in an attenuation of the fluorescence emission is observed upon the formation of mismatched base pairs.