To assess the effects of Ile359to Leu359change on CYP2C9-mediated metabolism, we performed site-directed mutagenesis and cDNA expression in yeast for CYP2C9 and examined in detail the kinetics of seven metabolic reactions by wild-type CYP2C9 (Ile359) and its Leu359variant. For the metabolism of all the substrates studied, the Leu359variant exhibited smallerVmax/Kmvalues than did the wild-type. The differences in theVmax/Kmvalues between the wild-type and the Leu359variant varied from 3.4-fold to 26.9-fold. The Leu359variant had higherKmvalues than did the wild-type for all the reactions studied. Among the seven reactions studied, the greatest difference in theVmaxvalues between the wild-type and the Leu359variant was for piroxicam 5′-hydroxylation (408 versus 19 pmol/min/nmol P450), whereas there were no differences in theVmaxvalues between the wild-type and the Leu359variant for diclofenac 4′-hydroxylation and tolbutamide methylhydroxylation. These results indicate that the Ile359to Leu359change significantly decreases the catalytic activity of all the CYP2C9-mediated metabolisms studied, whereas the extent of the reduction in activity and changes of the kinetic parameters varies between substrates. Moreover, the amino acid substitution decreased the enantiomeric excess in the formation of 5-(4-hydroxyphenyl)-5-phenylhydantoin from phenytoin.