Abstract: Thymidylate kinase catalyzes the phosphorylation of thymidine 5'-monophosphate to thymidine 5'-diphosphate in the pathway of synthesis of dTTP from dTMP. We have purified the enzyme approximately 5000-fold from a plasmid-bearing strain of the yeast Saccharomyces cerevisiae that over produces the activity 6-fold. The protein appears homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis and has a molecular weight of 25,000. The amino acid composition and the sequence of amino acids on the NH2 terminus have been determined. Our interest in thymidylate kinase stems from the fact that R. A. Sclafani and W. Fangman (personal communication) recently presented genetic evidence that this enzyme is encoded by the CDC8 gene of yeast. In this paper, we show, by several biochemical criteria, that thymidylate kinase is the product of the CDC8 gene. First, extracts of strains bearing six different alleles of cdc8 show no thymidylate kinase activity. Secondly, strains carrying the CDC8 gene on a high-copy-number plasmid produce 6-fold higher levels of the kinase activity than does wild type. Third, the DNA sequence of the CDC8 gene reveals an open reading frame that encodes a protein with the same amino-terminal sequence as purified thymidylate kinase.