5-Methyluridine (5MU) was synthesized efficiently from adenosine, thymine, and phosphate by a combination of adenosine deaminase (ADA), purine nucleoside phosphorylase (PUNP), pyrimidine nucleoside phosphorylase (PYNP), and xanthine oxidase (XOD). Adenosine was converted into inosine first by ADA. 5MU and hypoxanthine were synthesized from inosine and thymine by PUNP and PYNP. The hypoxanthine formed was converted into urate via xanthine by XOD. After inosine was completely consumed, an equilibrium state, in which 5MU, thymine, ribose-1-phosphate, and phosphate were involved, was achieved. At the equilibrium state, the maximum yield of 5MU was obtained. The yield of 5MU was 74%, when the initial concentrations of adenosine, thymine, and phosphate were 5 mM each. On the other hand, in the absence of ADA or XOD the yield of 5MU was 1.8%. Several kinds of nucleosides were also synthesized with high yield by the same method.