In the presence of ribothymidine, the uptake of 5-fluorouracil (5-FU) was enhanced in Ehrlich ascites carcinoma cells in vitro, and was efficiently converted to FU-nucleotides. In these cells, almost all ribothymidine was found to be converted to thymine by, presumably, uridine phosphorylase. The enhanced uptake of 5-FU and its efficient conversion to FU-nucleotides were also observed in Ehrlich ascites carcinoma of ribothymidine-coadministered mice. The radioactivity of 5-FU-¹⁴C in the carcinoma cells, which was detected mainly in the acid-soluble fraction immediately after the administration and then shifted to the ribonucleic acid fraction, was approximately 2-fold higher in the ribothymidine-coadministered group than in the control group. On the other hand, ribothymidine-¹⁴C was also taken up by the carcinoma cells and an appreciable protion of its radioactivity appeared in the thymine fraction. Thymine produced from ribothymidine-¹⁴C was also detected in the extracellular ascitic fluid. The conversion of ribothymidine to thymine was assumed to be catalyzed by uridine phosphorylase, suggesting that ribose 1-phosphate was coproduced and served as a ribose donor for 5-FU. These results suggest that ribothymidine may act as a ribose donor for 5-FU to form FU-nucleotides via 5-fluorouridine in carcinoma cells, resulting in the potentiation of 5-FU activity.