Nitrating reactive nitric oxygen species (RNOS) elicit many of the deleterious effects of the inflammatory response. Their high reactivity and short half-life make RNOS analysis difficult. Reaction of acetaminophen (APAP) with RNOS generated by various conditions was evaluated by HPLC. When [14C]APAP was incubated at pH 7.4, the same new product (3NAP) was produced by at least three separate pathways represented by the following conditions: myeloperoxidase oxidation of NO2-, NO2Cl, and ONOO- or Sin-1. Diethylamine NONO and spermine NONO did not convert APAP to 3NAP. 3NAP was stable at pH 5, 7.4, or 9, and at pH 7.4 with ONOO-, spermine NONO, Sin-1, or H2O2. HOCl transformed 3NAP, which was prevented by APAP, ascorbic acid, taurine, or NO2-. ONOO--derived 3NAP was identified by 1H NMR as 3-nitroacetaminophen or 3-nitro-N-acetyl-p-aminophenol, and the product mass was verified by EI/ESI mass spectrometry. Human polymorphonuclear neutrophils incubated with [14C]APAP and stimulated with β-phorbol 12-myristate 13-acetate produced 3NAP in the presence of NO2-. Neutrophil 3NAP formation was verified by mass spectrometry and was consistent with myeloperoxidase oxidation of NO2-. Spermine NONO supported 3NAP formation by stimulated cells in the absence of NO2-. Results demonstrate that 3NAP is a product of nitrating RNOS generated by at least three separate pathways and may be a biomarker for nitrating mediators of inflammation.