Acyl glucuronide conjugates are chemically reactive metabolites which can undergo hydrolysis, rearrangement (isomerization via acyl migration) and covalent binding reactions with protein. The present study was undertaken to identify factors modulating the reactivity of diflunisal acyl glucuronide (DAG) with human serum albumin (HSA) in vitro, by comprehensively evaluating the interplay of the three pathways above when DAG and a mixture of its 2-, 3- and 4-isomers (iso-DAG) were incubated with protein. Buffer, plasma, fraction V HSA, fatty acid-free HSA, globulin-free HSA and fatty acid- and globulin-free HSA were investigated at pH 7.4 and 37 degrees, each in the absence and presence of warfarin, diazepam and diflunisal (DF) as reversible binding competitors. DAG and iso-DAG were highly reversibly bound (ca. 98-99.5%) in plasma and HSA solutions. The binding was primarily at the benzodiazepine site, since displacement occurred in the presence of diazepam and fatty acids but not warfarin. DAG degradation, via rearrangement, hydrolysis and covalent adduct formation (in that order of quantitative importance), was retarded in plasma and HSA solutions compared to buffer. The protective effect of protein was afforded by the high reversible binding to the (non-catalytic) benzodiazepine site. The warfarin site appeared to be catalytic for DAG hydrolysis, whereas rearrangement appeared to be hydroxide ion-catalysed only. In contrast to DAG, iso-DAG degradation was greatly accelerated in the presence of protein, through both covalent binding and catalysis of hydrolysis. Covalent binding via DAG was increased in the presence of warfarin but decreased in the presence of diazepam, DF and fatty acids. The opposite effects were found for covalent binding via iso-DAG. The data suggest that covalent binding of DF to HSA via DAG and iso-DAG occurs by different mechanisms (presumably transacylation and glycation, respectively) at different sites (benzodiazepine and warfarin, respectively) whereas reversible binding occurs primarily at the same site (benzodiazepine).