Clozapine, a dibenzodiazepine antipsychotic, is associated with a 0.8% incidence of agranulocytosis. This clinically restrictive toxicity has been attributed to its chemically reactive metabolites. The generation of such metabolites--assessed via covalent binding and formation of thioether adducts--was investigated using human, rat and mouse liver microsomes and human neutrophils and bone marrow cells. In every instance, one major glutathione adduct of clozapine--C-6 glutathionyl clozapine--was formed in the presence of added glutathione. Adduct formation by the neutrophils and myeloid cells was dependent on cell activation by phorbol myristate acetate. Small fractions of drug underwent covalent binding to microsomes (1-6.8%) and to protein coincubated with neutrophils (0.47%) and myeloid cells (0.21%). Clozapine did not deplete intracellular glutathione in activated neutrophils. Clozapine was also metabolized in vivo to glutathione conjugates in rats and mice, the conjugates eliminated in bile over a 3-hr period representing 38% and 33% of the dose, respectively. In addition to the principal clozapine adduct found in vitro, the C-8 glutathionyl derivative of deschloroclozapine was excreted by both species. It is concluded that clozapine undergoes bioactivation in several tissues and considerable bioactivation in vivo. The reactive metabolites generated by neutrophils and myeloid cells may play an important role in the metabolic causation of clozapine-induced agranuiocytosis.