CYP2C9 catalyses the biotransformation of the oral anticoagulants S-warfarin and R- and S-acenocoumarol. According to data obtained in vitro, phenprocoumon is also metabolized by CYP2C9 but the impact of the CYP2C9 polymorphism on phenprocoumon pharmacokinetics has not been studied. Twenty-six healthy heterozygous and homozygous carriers of the CYP2C9 alleles *1 (wild-type), *2 (Arg144Cys), and *3 (Ile359Leu) received a single oral dose of 12 mg of racemic phenprocoumon. Plasma and 12-h urine concentrations of both enantiomers and their monohydroxylated metabolites were measured by high-performance liquid chromatography with mass spectrometry detection. No significant effect of the CYP2C9 variants *2 and *3 on R-phenprocoumon pharmacokinetic parameters was detected, but S-phenprocoumon clearance tended to decrease with increasing number of CYP2C9*2 and *3 alleles. The ratios of S- to R-phenprocoumon plasma clearances were higher with a median of 0.95 in carriers of *1/*1 versus 0.65 in *3/*3 (P < 0.001 for trend). Plasma and urine concentrations of 4′-, 6- and 7-hydroxyphenprocoumon were significantly lower in homozygous carriers of the CYP2C9*2 and *3 variants compared to CYP2C9*1/*1. Carriers of CYP2C9*3/*3 had a median AUC of (R,S) 7-OH-phenprocoumon of only approximately 25% compared to the wild-type genotype. The AUC of (R,S) 6-OH-phenprocoumon was only approximately 50% in CYP2C9*3/*3 compared to the homozygous wild-type genotype. In conclusion, carriers of CYP2C9*2 and *3 alleles had a lower metabolic capacity regarding phenprocoumon hydroxylation than those with CYP2C9*1/*1. However, regarding phenprocoumon hydroxylation CYP2C9 genotypes had only marginal effects on S- and R-phenprocoumon total clearance in healthy volunteers.