Previous studies have demonstrated that antibodies in sera from patients with halothane hepatitis recognize halothane-induced liver microsomal polypeptide neoantigens, and have suggested that these antibodies may play a role in the pathogenesis of the hepatitis. In the present study, the mechanism of neoantigen generation was investigated. Liver microsomes from rats treated in vivo with halothane or deuterated halothane were tested by immunoblotting for reactivity with patients' sera and with an antiserum specific for the covalently bound trifluoroacetyl (TFA) halide metabolite of halothane. Rat liver microsomes incubated aerobically or anaerobically with halothane or deuterated halothane in vitro, +/- NADPH and/or NADH, were also analyzed. The results obtained demonstrate that neoantigen expression involves oxidative halothane metabolism by cytochromes P-450 to TFA halide and covalent binding of the TFA group to the proteins. Incubation of microsomes from halothane-treated rats with 1 M piperidine cleaved the TFA groups from the proteins and abolished antigenicity, confirming this conclusion. Recognition of the neoantigens by the patients' antibodies was inhibited only partially using the hapten derivative N-E-TFA-L-lysine. It appears that the patients' antibodies recognize epitopes consisting of the TFA group plus associated structural features of the protein carriers (100 kDa, 76 kDa, 59 kDa, 57 kDa and 54 kDa), not the TFA hapten alone. To our knowledge, this constitutes the first characterization of drug metabolite-tissue protein neoantigens implicated in a drug hypersensitivity. The approach described may be of general utility for characterization of drug-induced neoantigens associated with other drug hypersensitivities.