The urine of mice injected intraperitoneally with pyrene during exposure to NO2 was found to contain highly mutagenic compounds by means of the Ames test using Salmonella typhimurium strain TA98. The mice were exposed to 20 ppm NO2 for 3 days before intraperitoneal injection of pyrene (800 mg/kg of body weight). The pyrene-treated mice were further exposed to NO2 for an additional 24 hr, and the urine from the mice was collected in ice-cooled containers and stored frozen in the dark. The collected samples were treated with beta-glucuronidase and passed through activated Sep-Pack C18 cartridges. After elution with methanol, the effluent was concentrated and the residue was dissolved in dimethyl sulfoxide (DMSO). The DMSO solution was fractionated by high-performance liquid chromatography and the mutagenicity of each fraction was assayed with S. typhimurium strain TA98. The mutagenic compounds 3-hydroxy-1-nitropyrene, 6-hydroxy-1-nitropyrene, 8-hydroxy-1-nitropyrene, and 1-hydroxypyrene were identified in the mutagenic fractions by mass spectrometry and UV-visible spectrophotometry with synthetic reference substances. These mutagenic compounds may have been formed by either nitration of hydroxylated pyrene, or hydroxylation of 1-nitropyrene, which is formed in vivo from pyrene and NO2, or the simultaneous occurrence of these two reactions in the mouse body.