Carboxylate drugs usually form acyl glucuronide conjugates as major metabolites. These electrophilic metabolites are reactive, capable of undergoing hydrolysis, rearrangement, and covalent binding reactions to proteins. The last-mentioned property has the potential to initiate immune and other toxic responses in vivo. In this study, we compared the extent and pattern of covalent adduct formation in plasma and livers of rats dosed with the nonsteroidal anti-inflammatory drugs (NSAIDs) zomepirac (ZP) and diflunisal (DF), the hypolipidemic agent clofibric acid (CA), and the anti-epileptic agent valproic acid (VPA). These drugs form acyl glucuronides with diverse intrinsic reactivities (apparent first order degradation t 1/2 values of 0.5, 0.6, 3, and 60 h, respectively). Rats were dosed iv twice daily for 2 days (50 mg/kg for ZP, DF, and CA, 150 mg/kg for VPA). Chemical analysis of tissues obtained 6 h after the last dose revealed adduct concentrations of 0.31, 0.44, 0.28, and 0.05 micrograms of drug equivalents/mL of plasma and 2.21, 2.31, 0.96, and 0.96 micrograms of drug equivalents/g of liver for ZP, DF, CA and VPA treatments, respectively. For both plasma and liver, the higher concentrations of adducts were found with ZP and DF, which have the more reactive glucuronides. The low concentrations of VPA adducts found in plasma were in keeping with the very low reactivity of its glucuronide. In liver, however, VPA adducts achieved concentrations of the same order of magnitude as the other drugs and were accompanied by adducts of the (E)-2-en metabolite of VPA at 0.38 micrograms of VPA equivalents/g of liver. The liver data for VPA can be explained by an acyl CoA/beta-oxidation pathway of adduct formation in addition to that from acyl glucuronidation. Immunoblotting using rabbit polyclonal antisera raised against synthetic drug-protein adducts revealed major bands at 110, 140, and approximately 200 kDa in livers of ZP- and DF-treated rats. A fourth major band at 70 kDa in ZP-treated liver had the same apparent molecular weight as the only major band detected in CA-treated liver. A 140 kDa band was detected in liver tissue from VPA-treated rats, as well as several lower molecular weight bands. In plasma, the antisera specifically detected drug-modified serum albumin in samples from rats treated with ZP, DF, and CA, but not VPA. The results with this small series of carboxylate drugs suggested that (a) adduct concentrations in plasma but not liver could be related to acyl glucuronide reactivity, (b) while some modified proteins detected were common, the pattern of modification varied from drug to drug, and (c) caution should be exercised in attributing adduct formation exclusively to the acyl glucuronidation pathway.