Carboxychroman metabolites of the major dietary tocopherols are excreted in human urine, but the mechanism of their synthesis is unknown. We employed well-characterized inhibitors of specific cytochrome P-450 (CYP) enzymes to determine which form was likely involved in tocopherol side chain oxidation. Ketoconozole (1.0 muM), a potent and selective inhibitor of CYP3A, substantially inhibited metabolism of gamma- and cu-tocopherol in rat primary hepatocytes, and metabolism of gamma- and delta -tocopherol in HepG2/C3A cells. Sulphaphenazole and cyclosporin, inhibitors of CYP2C and CYP27, respectively, were without effect. Sesamin, a sesame lignan that causes elevation of tissue tocopherol concentration in rats, strongly inhibited tocopherol metabolism by HepG2/C3A cells at 1.0 muM These results support a CYP3A-dependent mechanism of side chain metabolism of tocopherols to water-soluble carboxychromans, and provide the first evidence of a specific enzyme involved in vitamin E metabolism. The data further suggest that sesamin increases tissue tocopherol concentration by inhibiting tocopherol catabolism.