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The mechanism by which diclofenac-induced hepatotoxicity occurs is unclear, even though covalent modification of proteins by diclofenac metabolites appears to be important in pathogenesis, either by altering protein function or by eliciting an immune response. Adduct formation may be due to metabolism of diclofenac via an alternative pathway rather than via its major 4′-hydroxylation pathway mediated by the cytochrome P450 CYP2C9. We hypothesized that possession of variant CYP2C9 alleles might be a risk factor for diclofenac-induced hepatotoxicity, since the allelic variants CYP2C9*2 and CYP2C9*3 may be associated with impaired metabolism compared to the wild-type (CYP2C9*1). To investigate in more detail the effects of the polymorphisms on diclofenac metabolism in human liver, the kinetics of diclofenac 4-hydroxylation by human liver microsomes of known CYP2C9 >genotype were examined. An overall difference in Vmax and Vmax/ Km between samples homozygous for CYP2C9*1 and heterozygous for CYP2C9*2 or CYP2C9*3 was detected (P = 0.044). However, on subgroup analysis, there was no significant difference between samples homozygous for CYP2C9*1 and heterozygous for CYP2C9*2, although there was a borderline difference between the samples homozygous for CYP2C9*1 and those heterozygous for CYP2C9*3 (P = 0.057). The relationship between CYP2C9 genotype and susceptibility to diclofenac-induced hepatotoxicity was further examined by genotyping 24 patients with diclofenac-induced hepatotoxicity together with 100 healthy controls for the CYP2C9*2 and CYP2C9*3 alleles. CYP2C9 genotype frequencies for CYP2C9*2 and CYP2C9*3 were similar in patients and controls. To assess whether diclofenac-induced hepatotoxicity was due to rare CYP2C9 mutations, the upstream sequence (− 1 to − 1000) and all exons and exon–intron boundaries of CYP2C9 from four subjects who had suffered severe hepatotoxicity was determined. However, no new polymorphisms were detected. We therefore found no evidence that polymorphism in CYP2C9 is a determinant of diclofenac-induced hepatotoxicity.