We describe a method for distinguishing between minor groove binders and base intercalators that is based on measurements of the fluorescence lifetime of a donor (D) in the presence of an acceptor (A). The D–A pair is separated by a short double helix DNA with which the ligands interact. By plotting the D fluorescence lifetime as a function of the ligand-to-base pair concentration ratio we find a clear signature that distinguishes between the two binding mechanisms: minor groove binding induces an asymptotic decrease of the D fluorescence lifetime, while intercalation gives a monotonically increasing lifetime and the appearance of an additional short lifetime. We assayed Quinacrine, Hoechst and 4′–6′diamidine-2-phenyl indole, which in control experiments performed on oligodeoxyribonucleotides (oligos) lacking the A are demonstrated not to interfere with the D fluorescence. The changes in fluorescence lifetimes measured in the case of dual-labeled oligos are thus caused by structural changes in the DNA that modify the D–A distance. The appearance of the short-lived transient in the fluorescence decay of Ds attached to dual-labeled oligos upon binding of an intercalator can be interpreted as denaturation.