Purification and immunoinhibition studies have suggested that the hydroxylations of (S)-mephenytoin and tolbutamide are catalyzed by rather similar forms of human liver cytochrome P-450 (P-450). However, the two activities are not well correlated in vivo; sulfaphenzaole is a selective inhibitor of tolbutamide hydroxylation, and expression of P-450 2C10 cDNA in yeast yields a protein that hydroxylates tolbutamide but not (S)-mephenytoin. The P-450 2C8, 2C9, and 2C10 cDNAs have all been isolated, and their sequences are known to be closely related (greater than 80%). Highly sensitive radiochromatographic assays were set up, and tolbutamide and (S)-mephenytoin hydroxylation activities were monitored during chromatography of human liver microsomal fractions. The two activities could be separated by chromatography, and proteins were purified to near-homogeneity that catalyzed either tolbutamide hydroxylation (P-450TB) or (S)-mephenytoin 4'-hydroxylation (P-450MP) but not both. Approximately 16 and 45% of the primary sequences of P-450TB and P-450MP, respectively, were determined by analysis of the tryptic peptides. The sequences of the P-450TB peptides matched those predicted by the P-450 2C9 and 2C10 cDNAs exactly; the P-450MP peptides showed two mismatches (of 219 residues) with the P-450 2C10 sequence. Proteins with the P-450 2C10 and P-450 2C9 sequences were expressed in Saccharomyces cerevisiae grown under different nutritional conditions, and both were found to be proficient in the hydroxylation of tolbutamide but not (S)-mephenytoin. We conclude, on the basis of this and previous work, that 1) P-450s 2C8, 2C9, and 2C10 all catalyze the hydroxylation of tolbutamide and 2) the protein involved in polymorphic (S)-mephenytoin 4'-hydroxylation is closely related to but distinct from P-450 2C8, 2C9, and 2C10.