Use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with serious idiosyncratic hepatotoxicity. Covalent binding of reactive intermediates of DF to proteins is considered to initiate the process leading to this severe side-effect. The aim of this study was to characterize the nature of covalent protein modifications by reactive metabolites of DF which result from bioactivation by cytochrome P450. DF and its major monohydroxylated metabolites 4'-hydroxydiclofenac (4'-OH-DF) and 5-hydroxydiclofenac (5-OH-DF) were bioactivated using a highly active P450 BM3 mutant (CYP102A1M11H) in the presence of the model target protein human glutathione-S-transferase P1-1 (hGST P1-1). Protein-adducts were subsequently identified by LC-MS/MS analysis of tryptic digests of hGST P1-1. In total, 10 different peptide adducts were observed which result from modifications of Cys-47 and Cys-14 of hGST P1-1. The majority of the protein thiol modifications appeared to be derived from 5-OH-DF, which produced seven different peptide adducts with mass increments of 289.0, 309.0, and 339.0 Da. Remarkably, no peptide adducts were observed upon the bioactivation of 4'-OH-DF. Incubations of P450 BM3 with DF also showed the peptide adducts derived from 5-OH-DF and peptide adducts that are not derived from quinone imine. A peptide adduct with a mass increment of 249.0 Da most likely results from the o-imine methide formed by oxidative decarboxylation of DF. In addition, a peptide adduct was observed with a mass increment of 259.0 Da, which corresponds to the substitution of one of the chlorine atoms of DF by protein thiol. A corresponding GSH-conjugate with a similar mass increment was only observed if incubations of DF with P450 and GSH were supplemented by human GST P1-1. The results of this study not only confirm the importance of 5-OH-DF in covalent protein-binding but also suggest that the nature of protein adduction is not necessarily reflected by chemical conjugation with GSH.