Uridine/cytidine kinase (UCK), an important catalyst in the nucleotides replenishment pathway, which is responsible for the conversion of uridine and cytidine to 5'-uridine monophosphate (UMP) and 5'-cytidine monophosphate (CMP). UCK from Thermus thermophilus HB8 and Bacillus subtilis 168 was successfully cloned and expressed in Escherichia coli BL21 (DE3) respectively. The catalytic properties of different recombinant UCK were compared. Guanosine 5`-triphosphate (GTP) and adenosine 5`-triphosphate (ATP) was the main phosphate donor of UCK from Thermus thermophilus HB8, while GTP was the main phosphate donor of UCK from Bacillus subtilis 168. Under the optimized reaction conditions, 8.74 mM UMP was obtained from 10 mM uridine and 10 mM ATP in 6 h by the UCK from Thermus thermophilus HB8. The results showed that, the ATP-dependent UCK can be used in the research and production of UMP as a new catalyst.