To evaluate whether hepatocellular protein adducts of the nonsteroidal antiinflammatory drug diclofenac could elicit a specific cell-mediated or antibody-dependent immune response that eventually results in liver cell destruction, we developed a murine ex vivo/in vitro mixed lymphocyte hepatocyte culture (MLHC) model. C57BL/6 mice were immunized either with diclofenac conjugated to keyhole limpet hemocyanin (KLH) or with KLH alone. Splenocytes from mice exhibiting high anti-diclofenac antibody titers were isolated and co-cultured at an effector/target cell ratio of 100:1 with syngeneic murine hepatocytes preexposed to diclofenac. By 48 and 72 hours, extracellular alanine transaminase (ALT) activity had increased 6.4–and 7.6–fold, respectively, versus the 24-hour value. In contrast, there was no significant cytotoxic response after either drug treatment alone or immunization alone. Furthermore, those cellular populations capable of inducing ALT release also showed lymphocyte stimulation as determined by interleukin-2 (IL-2) receptor expression and lymphocyte proliferation analysis. The extent of cell injury was highest in the presence of lymphocytes highly enriched in T cells and was reduced by 40% in the presence of anti-MHC I antibodies. Similarly, albeit to a lesser extent, non-T cell-enriched lymphocyte fractions also induced hepatocyte injury. The addition of co-culture supernatants to hepatocytes had no effect, thus ruling out the possibility that soluble factors alone mediated the cell injury. However, supernatants from diclofenac-stimulated lymphocytes, combined with nonstimulated splenocytes, triggered an immediate (<1 hour) cytotoxic response, suggesting antibody-dependent cell-mediated mechanisms of target cell injury. These results indicate that diclofenac-treated hepatocytes carried antigenic determinants that were recognized by T cells and non-T cells derived from diclofenac/KLH-immunized mice, resulting in cell-mediated destruction of the target hepatocytes. (HEPATOLOGY 1995; 22:213–222.)