Thymidylate kinase is the product of the CDC8 gene of Saccharomyces cerevisiae (Jong, A.Y.S., Kuo, C.-L., and Campbell, J.L. (1984) J. Biol. Chem. 259, 11052-11059). In this communication we report the catalytic properties of the enzyme. The enzyme catalyzes the phosphorylation of deoxythymidine monophosphate to form deoxythymidine diphosphate in the presence of phosphate donor. ATP and dATP are the most efficient phosphate donors. In addition to dTMP, the yeast enzyme can use dUMP and 5-iodo-dUMP as phosphate acceptors. Kinetic analysis gives a Km of 0.5 mM for dTMP and 2 mM for dUMP. dTMP has a 7-fold greater rate constant than dUMP. Thymidylate kinase requires a divalent cation and is active over the entire range of pH from 6 to 9. The relative inhibitory effects of related compounds on yeast thymidylate kinase activity are in the order of dTDP greater than thymidine greater than 5-iodo-dUMP greater than ADP greater than or equal to dADP greater than dUMP greater than dTTP greater than dUDP, if dTMP is used as the phosphate acceptor. dTDP is a competitive inhibitor, with a Ki of 0.62 mM. Subcellular fractionation indicates that thymidylate kinase is found in the combined nuclear and cytoplasmic fraction but not in the mitochondria.