The effect of uracil on the metabolism of 5-fluorouracil (5-FU) in vitro was studied. 5-FU was mainly phosphorylated in intact Yoshida sarcoma cells, whereas it was mainly degraded in liver slices. Uracil inhibited degradation of 5-FU much more than its phosphorylation; incubation of 2,500 microM of uracil with 2.5 microM of 5-FU (molar ratio, 1,000:1) inhibited the degradation of 5-FU by 70%, but did not affect its phosphorylation. With homogenates of Yoshida sarcoma or liver uracil inhibited degradation of 5-FU greatly, phosphorylation of 5-FU by alpha-D-ribose 1-phosphate (RiblP) and ATP to some extent, and phosphorylation by 5-phospho-alpha-D-ribosyl diphosphate (PPRibP) very little. The activities of the enzymes involved in the metabolism of 5-FU in various tissues were also determined. Degradation of 5-FU was much faster in liver than in other tissues and was very slow in tumor tissue. Phosphorylation of 5-FU with RiblP and ATP was rapid in Yoshida sarcoma and bone marrow. Phosphoribosyltransferase activity was high in Yoshida sarcoma and thymus, but low in bone marrow.