Acyl glucuronide metabolites of carboxylic acid drugs such as the salicylate derivative diflunisal (DF) have been shown to react with proteins in vitro and in vivo to produce covalent adducts. Such attachment of foreign compounds to endogenous molecules could be associated with toxic and/or immune consequences. In this study we have injected rats with rat serum albumin (RSA) modified (a) by DF using a carbodiimide reagent (-->DF-RSA-I, 4.9 micrograms DF/mg RSA) and (b) by incubation with DF acyl glucuronide (DAG) and its rearrangement isomers (iso-DAG) (-->DF-RSA-II, 0.34 micrograms DF/mg RSA). All of the six rats injected with DF-RSA-I produced antibodies reactive with DF-modified keyhole limpet hemocyanin (KLH), the coating protein used in the ELISA. Three out of six animals injected with DF-RSA-II generated similar antibodies. Cross-reactivity with other non-steroidal anti-inflammatory drugs (NSAIDs) such as naproxen and ketoprofen (as the free drugs) was not observed. This study shows that a self protein covalently modified by incubation with DAG and iso-DAG is immunogenic in rats. The data thus support the hypothesis that covalent modification of macromolecules by acyl glucuronide metabolites of acidic drugs in vivo can lead to the production of circulating antibodies which may be involved in aberrant immune responses such as drug hypersensitivity.