The non-steroidal anti-inflammatory drug diclofenac is reported to cause, in rare cases, fulminant hepatic necrosis associated with chronic use of the drug. In order to investigate the possibility that covalent protein adducts of reactive metabolites of diclofenac might be responsible for the hepatotoxicity produced by this drug, we developed an antibody against a diclofenac-keyhole limpet hemocyanin adduct. The anti-diclofenac antibody did recognize diclofenac-protein adducts on Western blots of homogenates of cultured human hepatocytes exposed to diclofenac. The major detected adduct was a 60 kDa protein, which was present in both human and rat hepatocytes. These results suggest that binding of diclofenac to human hepatocytes is, like in rats, selective and that a 60 kDa protein appears to be the major target for alkylation. Immunoblots of homogenates of liver sinusoidal lining cells (LSLC) from rats treated with diclofenac also exhibited adducts with a 60 kDa protein. This fact suggest a role for LSLC in processing of chemically altered proteins in the liver.