Previous investigations have implicated an immune response to trifluoroacetylated proteins (TFA-proteins) in the pathogenesis of halothane hepatitis. The objective of this study was to establish conditions for generation of TFA-proteins in hepatocytes exposed to halothane in vitro. Monolayer cultures of rat hepatocytes were incubated in sealed flasks with or without added halothane, then subcellular fractions were prepared by differential centrifugation and analysed by immunoblotting for reactivity with anti-TFA antiserum. The specificity of the antiserum was verified by hapten inhibition with N--TFA-l-lysine. TFA-proteins were generated when hepatocytes were cultured with halothane, but not when hepatocytes were cultured without halothane, and were concentrated in the microsomal fraction. Generation of TFA-proteins was greater when hepatocytes were exposed to an initial halothane concentration of about 0.17 mm-halothane than when hepatocytes were exposed to higher concentrations (0.6 mm and 1.4 mm). The molecular masses of the major TFA-proteins produced in vitro (100, 80 and 60 kDa) were very similar, if not identical, to the molecular masses of the major TFA-proteins produced in livers of rats treated ip with halothane in vivo, as were the kinetics of TFA-protein formation and turnover.