Clozapine was oxidized to a reactive intermediate by HOCI, which is the major oxidant produced by activated neutrophils. A mass spectrum was obtained of this reactive intermediate by using a flow system in which the reactants were fed into a mixing chamber and the products flowed directly into a Sciex API III mass spectrometer. The intermediate was observed at m/z 325, which is 2 mass units less than the protonated molecular ion of the parent drug. This intermediate reacted with water to form several products with a m/z at 343. The same products were produced by the oxidation of clozapine by the combination of myeloperoxidase, hydrogen peroxide and chloride ion. The reactive intermediate was trapped by glutathione (GSH) and several conjugates were formed. Nuclear magnetic resonance spectra of the two major conjugates indicated GSH bound to the 6 and 9 positions of the aromatic ring. These data provide further evidence for the formation of a formal nitrenium ion in which the positive charge is highly delocalized. Clozapine was also oxidized by activated neutrophils, and in the presence of GSH, the same GSH conjugates were formed. When therapeutic concentrations of radiolabeled clozapine were used, up to 7% of the drug became irreversibly bound to the neutrophils. Covalent binding was inhibited by about 30% in the presence of 1 mM GSH but was almost abolished at 5 mM GSH. The putative nitrenium ion formed by activated leukocytes could be responsible for clozapine-induced agranulocytosis.