Previous biochemical studies have suggested that tolbutamide and mephenytoin are metabolized by the same cytochrome P450 enzyme. Conversely, clinical studies indicate the involvement of different P450 forms in tolbutamide and mephenytoin metabolism. Our objective was to elucidate further those P450 enzymes responsible for hydroxylation of these two drugs. We studied both tolbutamide and (S)-mephenytoin hydroxylation in microsomes from 38 different normal adult human livers, and found large variability in the rates of metabolism for both reactions (1.75-47.4 nmol/mg/hr for hydroxytolbutamide formation and 0.1-7.2 nmol/mg/hr for 4-hydroxymephenytoin formation). No significant correlation was found between the two activities. However, both reactions shared common inhibitors in vitro, including inhibition by antikidney-liver-microsome autoantibodies (Meier and Meyer, Biochemistry 26: 8466-8474, 1987) and by teniposide. Two human liver cDNAs for P450s of the CYP2C subfamily designated IIC8 and IIC9 (S. Kimura, J. Pastewka, H. V. Gelboin and F. J. Gonzalez, Nucl. Acids Res. 15: 10053-10054, 1987), were functionally expressed in human HepG2 and TK- cells using a vaccinia virus vector. Interestingly, tolbutamide was hydroxylated by both expressed P450s. Only IIC9 catalyzed the 4-hydroxylation of (R)-mephenytoin and neither enzyme metabolized (S)-mephenytoin. We conclude that tolbutamide and (R)-mephenytoin are both metabolized by the same P450 enzyme, IIC9, and that tolbutamide is hydroxylated by an additional highly related enzyme, IIC8, contributing to the lack of correlation of the two hydroxylase activities among human liver microsomes and indicating the absence of a monogenically controlled polymorphism for tolbutamide.