Benoxaprofen (BNX) has been implicated in rare but serious hepatotoxicity which led to its withdrawal from the world market. Flunoxaprofen (FLX), a structural analog, appears to be less toxic. It has been postulated that the nonsteroidal antiinflammatory drugs associated toxicity may be related to covalent modification of proteins by their reactive acyl glucuronides, and the extent of covalent protein binding depends on both reactivity of the acyl glucuronide and the exposure to the reactive metabolite. The disposition of BNX and FLX in rats were compared upon intravenous administration of 20 mg/kg of BNX, FLX or their metabolites. Covalent binding of BNX and FLX to plasma and liver proteins were also determined, and an immunochemical approach was used to detect their hepatic targets. Similar concentrations of plasma protein adducts for BNX and FLX were detected even though the AUC of BNX-glucuronide (BNX-G) was almost twice that of FLX-glucuronide (FLX-G). Similar concentrations of liver protein adducts for BNX and FLX were also detected at 8 h, however, the hepatobiliary exposure of BNX-G was only 1/3rd that of FLX-G indicating that BNX-G was more reactive than FLX-G, which was in agreement with in vitro data. Proteins of 110 and 70 kDa were the major liver protein targets modified by covalent attachment of BNX and FLX. In conclusion, measuring covalent binding to tissue proteins in animals in addition to plasma adducts should be considered when evaluating and comparing carboxylic acid analogs that form reactive acyl glucuronides.