Clozapine, an atypical antipsychotic used in the treatment of refractory schizophrenia, causes neutropenia and agranulocytosis in 3 and 0.8% of patients, respectively. Clozapine undergoes bioactivation to a chemically reactive nitrenium ion, which has been shown to cause neutrophil cytotoxicity. To define further the mechanism of cell death, we have investigated the toxicity of clozapine, its stable metabolites, and its chemically reactive nitrenium ion to neutrophils and lymphocytes. Clozapine was able to induce neutrophil apoptosis at therapeutic concentrations (1-3 microM) only when it was bioactivated to the nitrenium ion. The parent drug caused apoptosis at supratherapeutic concentrations (100-300 microM) only. Neutrophil apoptosis induced by the nitrenium ion, but not by the parent drug itself, was inhibited by antioxidants and genistein and was accompanied by cell surface haptenation (assessed by flow cytometry) and glutathione depletion. Dual-color flow cytometry showed that neutrophils that were haptenated were the same cells that underwent apoptosis. No apoptosis of lymphocytes was evident with the nitrenium ion or the parent drug, despite the fact that the former caused cell surface haptenation, glutathione depletion, and loss of membrane integrity. Demethylclozapine, the major stable metabolite in vivo, showed a profile that was similar to, although less marked than that observed with clozapine. N-oxidation of clozapine or replacement of the nitrogen (at position 5) by sulfur produced compounds that were entirely nontoxic to neutrophils. In conclusion, the findings of the study expand on potential mechanisms of clozapine-induced cytotoxicity, which may be of relevance to the major forms of toxicity encountered in patients taking this drug.