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Fluorescently labeled oligonucleotides and DNA fragments have promise in nucleic acid research with applications that include DNA hybridization, automated DNA sequencing, fluorescence anisotropy, and resonance energy transfer studies. Past concerns with fluorescent-labeled DNA arose from interactions between fluorophores and DNA that result in quenched fluorescence. This quenching phenomenon is most problematic in fluorescence resonance energy transfer studies because quenching of the donor fluorescence could result from either resonance energy transfer or nontransfer effects. In the present study, relief of nontransfer quenching of a 14-mer fluorescein 5-isothiocyanate (FITC)-labeled oligonucleotide containing the BamHI restriction site was characterized with both steady-state and time-resolved fluorescence techniques. The FITC-labeled single strand was best fit by a triexponential decay with lifetimes of 0.5, 2.7, and 4.2 ns. The 4.2-ns component was found to contribute more than 80% of the total steady-state intensity. Upon annealing with an unmodified complementary strand, the contribution from the 4.2-ns component was significantly decreased, resulting in twofold quenching of total fluorescence. We reasoned that this quenching phenomenon should be a reversible process and could be employed to study strand separation processes in molecular biology. Hence, cleavage of the fluorescently labeled substrate was examined using DNAase I and BamHI restriction endonuclease. Our results show that the quenched fluorescence is totally recovered upon cleavage (compared to that of the single strand). The extent of cleavage measured by fluorescence was confirmed by nondenaturing polyacrylamide gel electrophoresis analysis. We believe this fluorescence "dequenching" technique may be used to quantify the kinetics of other DNA strand separation and cleavage processes in molecular biology.