We report the first demonstration of EPR spectroscopy to study free radical reactions in live mice and excised muscle tissue resulting from the metabolism of nitrosobenzene. A broad three-line EPR spectrum (aN = 11.6 G) appeared in the buttocks region of a mouse place in an L-band loop gap resonator after intramuscular or intraperitoneal injection of 0.2 mmol/kg nitrosobenzene. The signal intensity reached a maximum at 20 to 30 min and remained constant well beyond 2 h. If muscle tissue was dosed with nitrosobenzene and excised within 5 min, a similar three-line X-band EPR spectrum was obtained which was preceded by the rapid growth and subsequent decay of an EPR spectrum identical with that of the phenylhydronitroxide radical, which was presumably generated from reactions between nitrosobenzene and reducing agents in the blood or tissue such as NADH or ascorbic acid. A model system containing nitrosobenzene and unsaturated fatty acids (olive oil or animal fat) yielded an identical three-line spectrum resulting from radical adducts of nitrosobenzene across the double bond. Overall, these results suggest that the most probable mechanism in vivo was nitrosobenzene covalently adding ("binding") to polyunsaturated fatty acid clusters in fat or membranes.