The present study assesses the role of members of the human CYP2C subfamily in the 4'-hydroxylation of (S)-mephenytoin. When recombinant CYP2C proteins were expressed using a yeast cDNA expression system, 2C19 stereospecifically 4'-hydroxylated (S)-mephenytoin with a turnover number at least 10 times higher than that of human liver microsomes. 2C9 (both Ile359 and Leu359 alleles) and 2C18 (Thr385 and Met385 alleles) metabolized this substrate at a rate 100-fold lower than 2C19, and metabolism by these 2C proteins was not stereospecific for the S-enantiomer. 2C8 exhibited very little mephenytoin 4'-hydroxylase activity. In contrast, the Ile359 allele of 2C9 had a high turnover number for the hydroxylation of tolbutamide, while the Leu359 allele was less active toward this substrate. Immunoblot analysis of 16 human liver donor samples indicated that (S)-mephenytoin 4'-hydroxylase activity correlated with the hepatic CYP2C19 content, but it did not correlate with the hepatic content of CYP2C9. Moreover, direct sequencing of the polymerase chain reaction (PCR) products of 2C9 mRNA from six of these human livers through areas of known allelic variations indicated that the identity of the allele of 2C9 (Cys144 vs Arg, Tyr358 vs Cys, Ile359 vs Leu, or Gly417 vs Asp) did not appear to influence (S)-mephenytoin 4'-hydroxylase activity in these samples. These data indicate that 2C19 is the principal determinant of (S)-mephenytoin 4'-hydroxylase activity in human liver.