We hypothesized that measurement of a specific product from reaction of N,N'-dimethylthiourea (Me2TU) and H2O2 would provide a good indication of the H2O2 scavenging and protection seen after addition of Me2TU to biological systems. We found that addition of H2O2 to Me2TU yielded a single stable product, Me2TU dioxide. Me2TU dioxide formation correlated with Me2TU consumption as a function of added H2O2 concentration and was prevented by simultaneous addition of catalase (but not boiled catalase), superoxide dismutase, dimethyl sulfoxide, mannitol, or sodium benzoate. Me2TU dioxide formation, Me2TU consumption, and H2O2 concentration increases occurred in mixtures containing phorbol 12-myristate 13-acetate (PMA) and normal human neutrophils but not in mixtures containing PMA and neutrophils from patients with chronic granulomatous disease or in mixtures containing PMA and normal neutrophils and catalase. Me2TU dioxide formation also occurred in isolated rat lungs perfused with Me2TU and H2O2 but not in lungs perfused with Me2TU and elastase, histamine, or oleic acid. In contrast, Me2TU dioxide formation did not occur after exposure of Me2TU to 60Co-generated hydroxyl radical or hypochlorous acid in the presence of catalase. The results indicate that reaction of Me2TU with H2O2 selectively forms Me2TU dioxide and that measuring Me2TU dioxide formation from Me2TU may be useful for assessing the presence and significance of H2O2 in biological systems.