Arylamine-hemoglobin conjugates identified as sulfinamides are considered dosimeters for the bioavailability of metabolically formed N- oxidation products. This report considers peroxidation as an alternative pathway for aromatic amine metabolism and examines horseradish peroxidase metabolism of N-acetylbenzidine (ABZ) in the presence of glutathione. When 0.06 mM [3H]ABZ was incubated with 1 mM glutathione, a decrease in the total extent of metabolism was observed along with detection of a new metabolite (ABZ-SG), representing 12% of the total radioactivity. Optimum ABZ-SG formation occurred at 0.3 mM glutathione with higher concentrations (10 mM) being inhibitory. In the absence of glutathione, a molar ratio of H2O2 to ABZ of 1:1 resulted in complete metabolism of ABZ. This ratio increased to >2:1 in the presence of 0.3 mM glutathione. N-Oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetylbenzidine, were not detected using a variety of incubation conditions. ABZ-SG was sensitive to γ- glutamyltranspeptidase, and completely hydrolyzed by 0.1 N HC1 or 0.1 N NaOH in 10 min at room temperature. ABZ-SG was identified by mass spectrometry and NMR to be N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide. ABZ-SG formation, but not total ABZ metabolism, was prevented by 0.3 mM NaN3, 50 mM DMPO, 1.0 mM thiourea, and 1.0 mM histidine. Cyanide (50 mM) and ascorbic acid (0.1 mM) completely inhibited ABZ metabolism. The lack of effect of 50 mM mannitol and 2 μg of superoxide dismutase suggests that neither hydroxyl radical nor superoxide is involved in the reaction. Studies also indicated that molecular oxygen is not a source of the sulfinamide oxygen. Formation of an ABZ sulfinamide conjugate with hemoglobin was demonstrated. The proposed mechanism for sulfinamide formation, involving two consecutive one-electron oxidations with subsequent rearrangement to a sulfur-stabilized nitrenium ion, suggests that oxygen may be derived from water. The results demonstrate that while arylamine-hemoglobin conjugates serve as useful biomarkers of exposure, their mechanism of formation may be complex, perhaps involving peroxidation as in the case of N'-(glutathion-S-yl)-N-acetylbenzidine S- oxide.