Cytochrome P-450-dependent oxidative cleavage of 1-(tetrahydro-2-furanyl)-5-fluorouracil (FT) was investigated in a reconstituted system containing purified phenobarbital-inducible cytochrome P-450 (P-450(1)) or 3-methylcholanthrene-inducible cytochrome P-450 (P-448(1)). FT was converted into 5-fluorouracil (5-FU) in the reconstituted system, and its rate was 71 pmol 5-FU formed/min/nmol P-4501 and 45 pmol 5-FU/min/nmol P-448(1). Cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH were required for 5-FU production. Inhibitors of cytochrome P-450 such as carbon monoxide and metyrapone markedly decreased the rate. FT was found to interact with the purified cytochrome P-450, causing a reverse type I spectral change. From these observations, we concluded that the hepatic microsomal cytochrome P-450-dependent mixed function oxidase system participates in the oxidative cleavage of FT.