4-Substituted 2,6-dimethyl-3,5-bis(alkoxycarbonyl)-1,4-dihydropyridines are important because of their roles as calcium channel blockers. The mixed-function oxidation of 14 4-aryl- and four 4-alkyl-substituted derivatives by human liver microsomes was examined. The major product of enzymatic oxidation of all the 4-aryl compounds was the pyridine derivative containing the 4-aryl group. The 4-alkyl compounds, in contrast, formed a pyridine derivative in which a hydrogen atom was present at the 4-position and the alkyl group was lost; these compounds also inactivated cytochrome P-450 and caused the loss of nifedipine oxidase activity after enzymatic oxidation. All of these reactions were extensively inhibited by an antibody raised to purified human liver nifedipine oxidase cytochrome P-450 (P-450NF), indicating a major role for this enzyme in the oxidation of these compounds. Oxidation of the 4-alkyl compounds led not only to the loss of P-450NF but also to decreases in catalytic activities of cytochrome P-450 isozymes catalyzing other reactions (phenacetin O-deethylation and hexobarbital 3'-hydroxylation). The results indicate that P-450NF (or closely related enzyme forms) is responsible for the oxidation of these nifedipine-related compounds in human liver microsomes and that metabolism is highly dependent upon 4-substitution; with alkyl substituents, radicals are postulated to leave P-450NF to attack other proteins.