We present evidence herein of the accelerated generation of hydroxyl radical (.OH) in the plasma and the liver tissue of common bile duct ligated (CBDL) rats, a model for experimental obstructive jaundice. .OH production in the plasma was monitored in vivo by the identification of dihydroxybenzoates in plasma [2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (2,5-DHB)] using high performance liquid chromatography (HPLC). The average concentrations of 2,3-DHB and 2,5-DHB produced in the plasma of the controls were 33+/-3 microM and 232+/-34 microM (n = 15), respectively, whereas their respective concentrations increased to 149+/-28 microM and 604+/-88 microM in the CBDL rats (n = 19). Furthermore, we also observed a time-dependent decreasing trend of 2,3-DHB and 2,5-DHB production after surgical removal of the ligation of the experimental animals. In addition, the generation of .OH in the liver tissue was studied by using dimethyl sulfoxide (DMSO) as a molecular probe and measuring the amount of methanesulfinic acid (MSA), the product of the trapping reaction. The net production of MSA in the liver tissue of the control rats was 1.22+/-0.05 O.D. unit/g protein (n = 5), whereas its respective concentration of MSA in the liver tissue of CBDL rats increased to 2.05+/-0.15 O.D. unit/g protein (n = 5). In addition, we showed that CBDL rats receiving a pretreatment of mannitol, an .OH scavenger, resulted in the decreased production of MSA. Electron micrographic study indicated that the most prominent change observed in CBDL rats was the alteration of mitochondria, which were swollen with distorted cristae. Meanwhile, the bile canaliculi were moderately more dilated than that of the controls, and an increased neutrophil peripheral blood count was found in CBDL rats when compared to the controls. Taken together, our data suggest that accelerated generation of .OH in the CBDL rats is obvious and may play a key role in the pathogenesis of liver damage associated with obstructive jaundice.