Animal studies of therapeutic oligonucleotides require measurement of circulating levels of oligonucleotides by multistep, time-consuming methods. In contrast, addition of a single-stranded DNA binding fluorophore, OliGreen, to oligonucleotides in plasma samples allowed rapid quantitation. Dose-response curves were measured for five different oligonucleotide analogs added to plasma or serum. Phosphorothioate or 3'-amino phosphodiester oligodeoxynucleotides in calf serum reliably exhibited linear, dose-dependent fluorescence at 15-500 nM. The assay was equally sensitive in human and mouse plasma, with a heterogeneous variety of sequences. Oligonucleotides shorter than 10 nucleotides yielded substantially reduced fluorescence. In contrast, 2'-O-methyl oligoribonucleotides, DNA methylphosphonates, and peptide nucleic acids demonstrated little or no fluorescence with OliGreen. Following intravenous injection of a phosphorothioate pentadecamer into mice, fluorescence measurements of plasma phosphorothioate levels displayed a dose-dependent, biexponential decline over a 90 min period. Chronic infusion at 2.5 nmol/hour into mice yielded plasma oligonucleotide values equivalent to 0.1 microM, a value reflecting the contributions of intact and partially degraded strands. Tumor-bearing mouse plasma evidenced high fluorescence values in the absence of oligonucleotide administration, presumably because of elevated intrinsic plasma DNA fragments. Although limited in its ability to differentiate intact from partially degraded strands, OliGreen fluorescence provides a simple, rapid, and sensitive method for measuring circulating levels of phosphorothioate or phosphodiester oligonucleotides in healthy animals or humans.