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 variantsCYP2C9*2andCYP2C9*3may 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 knownCYP2C9>genotype were examined. An overall difference inVmaxandVmax/Kmbetween samples homozygous forCYP2C9*1and heterozygous forCYP2C9*2orCYP2C9*3was detected (P= 0.044). However, on subgroup analysis, there was no significant difference between samples homozygous forCYP2C9*1and heterozygous forCYP2C9*2, although there was a borderline difference between the samples homozygous forCYP2C9*1and those heterozygous forCYP2C9*3(P= 0.057). The relationship betweenCYP2C9genotype and susceptibility to diclofenac-induced hepatotoxicity was further examined by genotyping 24 patients with diclofenac-induced hepatotoxicity together with 100 healthy controls for theCYP2C9*2andCYP2C9*3alleles.CYP2C9genotype frequencies forCYP2C9*2andCYP2C9*3were 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 ofCYP2C9from four subjects who had suffered severe hepatotoxicity was determined. However, no new polymorphisms were detected. We therefore found no evidence that polymorphism inCYP2C9is a determinant of diclofenac-induced hepatotoxicity.