Administration of the carcinogen ethylene dibromide (EDB) to rats resulted in the urinary excretion of S-[2-(N7-guanyl)ethyl]-N-acetylcysteine, which is derived from the nucleic acid adduct S-[2-(N7-guanyl)ethyl]glutathione. This mercapturic acid was isolated from urine by reversed-phase and propylamino high-performance liquid chromatography and was quantitated by measurement of fluorescence intensity. The urinary mercapturic acid was identified as S-[2-(N7-guanyl)ethyl]-N-acetylcysteine on the basis of cochromatography and UV, fluorescence, 1H nuclear magnetic resonance, and fast atom bombardment mass spectra, all of which were identical with the authentic synthesized material. The excretion of mercapturic acid into urine of rats given injections of various doses of EDB occurred in a dose-dependent, linear manner over the range of 0.5-37 mg EDB/kg body weight. A good correlation was found between the excretion of mercapturic acid and the (in vivo) formation of DNA adducts in liver and kidney DNA. The higher level of urinary mercapturic acid compared to the level of hepatic DNA adduct indicates that extra-hepatic DNA adducts and RNA adducts may contribute to the mercapturic acid production. The measurement of the mercapturic acid may provide a means of noninvasive estimation of DNA adducts derived from EDB exposure.