Introduction: The polymorphic cytochrome P450 enzyme 2C9 (CYP2C9) catalyses the metabolism of many drugs including S-warfarin, acenocoumarol, phenytoin, tolbutamide, losartan and most of the non-steroidal anti-inflammatory drugs. Diclofenac is metabolised to 4′-hydroxy (OH), the major diclofenac metabolite, 3′-OH and 3′-OH-4′-methoxy metabolites by CYP2C9. The aim of the present study was to clarify the impact of the CYP2C9 polymorphism on the metabolism of diclofenac both in vivo and in vitro. Subjects, materials and methods: Twenty healthy volunteers with different CYP2C9 genotypes [i.e. CYP2C9*1/*1 (n=6), *1/*2 (n=3), *1/*3 (n=5), *2/*3 (n=4), *2/*2 (n=1), *3/*3 (n=1)] received a single 50-mg oral dose of diclofenac. Plasma pharmacokinetics [peak plasma concentration (Cmax), half-life (t1/2) and area under the plasma concentration–time curve (AUCtotal)] and urinary recovery of diclofenac and its metabolites were compared between the genotypes. Diclofenac 4′-hydroxylation was also analysed in vitro in 16 different samples of genotyped [i.e. CYP2C9*1/*1 (n=7), *1/*2 (n=2), *1/*3 (n=2), *2/*3 (n=2), *2/*2 (n=2), *3/*3 (n=1)] human liver microsomes. Results: Within each genotype group, a high variability was observed in kinetic parameters for diclofenac and 4′-OH-diclofenac (6- and 20-fold, respectively). No significant differences were found between the different genotypes either in vivo or in human liver microsomes. No correlation was found between the plasma AUC ratio of diclofenac/4′-OH-diclofenac and that of losartan/E-3174, previously determined in the same subjects. Conclusion: No relationship was found between the CYP2C9 genotype and the 4′-hydroxylation of diclofenac either in vivo or in vitro. This, together with the lack of correlation between losartan oxidation and diclofenac hydroxylation in vivo raises the question about the usefulness of diclofenac as a CYP2C9 probe.