The existence of multifunctional enzymes in the nucleotide biosynthesis pathways is believed to be one of the important mechanisms to facilitate the synthesis and the efficient supply of deoxyribonucleotides for DNA replication. Here, we used the bacterially expressed yeast thymidylate kinase (encoded by the CDC8 gene) to demonstrate that the purified Cdc8 protein possessed thymidylate-specific nucleoside diphosphate kinase activity in addition to thymidylate kinase activity. The yeast endogenous nucleoside diphosphate kinase is encoded by YNK1, which appears to be non-essential. Our results suggest that Cdc8 has dual enzyme activities and could duplicate the function of Ynk1 in thymidylate synthesis. We also discuss the importance of the coordinated expression of CDC8 during the cell cycle progression in yeast.