Fluorouracil (5-FU) prodrug tegafur (FT) is used widely for treating cancer patients. It has been reported that CYP2A6 and thymidine phosphorylase (TP) are involved in the formation of 5-FU from FT. In this study, the relative contribution of cytochrome P450 (P450) to the formation of 5-FU from FT was assessed using human liver 9000 x g supernatant (S9) and hepatocytes, which contain both enzymes. Intrinsic clearances of 5-FU formation from FT by P450 (NADPH dependent) and TP (NADPH independent) in human liver S9 were 1.36 and 0.169 microL/min/mg protein, respectively. The formation of 5-FU from FT in human liver S9 was inhibited over 82% by 8-methoxypsoralen, a CYP2A6-selective inhibitor. The formation of 5-FU from FT was also evaluated in human hepatocytes, cells that not only exhibit P450 and TP activity but also have anabolic capacity. The results indicated that CYP2A6 played a major role in 5-FU formation, which was consistent with the results using human liver S9. Factors that can affect the level of CYP2A6 activity in patients, e.g., genetic polymorphism, should be considered when using FT for chemotherapy.