8-Oxoguanine (8-oxo-G) is one of the most common DNA lesions present in normal tissues due to exposure to reactive oxygen species. Studies at this and other laboratories suggest that 8-oxo-G is highly susceptible to secondary oxidation, making it a likely target for endogenous oxidizing agents, such as peroxynitrite (ONOO-). Synthetic oligonucleotides containing 8-oxoguanine were treated with ONOO-, and the reaction products were analyzed by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI--MS). CCACAACXCAAA, CCAAAGGXAGCAG, CCAAAXGGAGCAG, and TCCCGAGCGGCCAAAGGXAGCAG (X is 8-oxo-G) were found to readily react with peroxynitrite via the same transformations as those observed for free 8-oxo- 2'-deoxyguanosine. The composition of the reaction mixtures was a function of ONOO- concentration and of the storage time after exposure. The oligonucleotide products isolated at low [ONOO-]/[DNA] ratios (<5) were tentatively assigned as containing 3a-hydroxy-5-imino-3,3a,4,5-tetrahydro- 1H-imidazo[4,5d]imidazol-2-one, 5-iminoimidazolidine-2,4-dione, and its hydrolytic product, oxaluric acid. At a [ONOO-]/[DNA] ratio of > 10, 2,4,6- trioxo[1,3,5]triazinane-1-carboxamidine- and cyanuric acid-containing oligomers were the major products. The exact location of a modified base within a DNA sequence was determined using exonuclease digestion of oligonucleotide products followed by LC/ESI--MS analysis of the fragments. For all 8-oxo-G-containing oligomers, independent of the sequence, the reactions with ONOO- took place at the 8-oxo-G residues. These results suggest that 8-oxo-G, if present in DNA, is rapidly oxidized by peroxynitrite and that oxaluric acid is a likely secondary oxidation product of 8-oxo-G under physiological conditions.