3-Nitrofluoranthene, a potent environmental mutagen, was administered to rats intraperitoneally at 10 mg/kg, labelled with14C. Urine contained 15 to 20% of the dose, eliminated mainly within the first 24 hours after dosing. Over 95% of the radioactivity in unhydrolysed urine chromatographed with the solvent front in a reverse-phase HPLC system, indicating extensive conjugation. After hydrolysis with β-glucuronidase (containing sulfatase) and concentration on C18-Sep-Pak, urine was fractionated by HPLC for further characterization. NMR analysis of the metabolite fractions indicated that positions 4, 8 and 9 were major sites of oxidation. Both acetamide and amine derivatives were formed. The finding of multiple oxidation and incomplete acetylation is in contrast to the fate of 1-nitropyrene, where 1-acetamidopyren-6-ol, itself a potent mutagen, represents the major metabolic product. This difference in metabolism pathways may have implications for the genotoxicity of 3-nitrofluoranthene.