Diclofenac has been used for the evaluation of CYP2C9 activity in vitro as well as in vivo with varying results. The present study was aimed at evaluating the reproducibility of the urinary diclofenac/4'-OH diclofenac ratio among different CYP2C9 genotypes in healthy volunteers. The study of CYP2C9 genotypes in the family of a CYP2C9*3/*3 subject is also reported. The urinary diclofenac/4'-OH diclofenac ratio was determined on two occasions within a period of 9-12 months, and was found to be correlated (r = 0.83, p < 0.05). The mean (+/- SD) of diclofenac/4'-OH diclofenac ratio was 1.5 times higher among subjects carrying CYP2C9*3 allele (CYP2C9*1/*3 and CYP2C9*2/*3 genotypes) (0.91 +/- 0.28), compared to CYP2C9*1/*1 subjects (0.60 +/- 0.11). The results show that the urinary diclofenac/4'-OH diclofenac ratio might be used to study CYP2C9 in humans. The data agree with previous studies showing that the CYP2C9*3 allelic variant seems to cause a decreased CYP2C9 hydroxylation capacity.