dTMP kinase (E.C.188.8.131.52.) catalyzes the phosphorylation of dTMP to the corresponding diphosphate. This enzyme is essential for DNA synthesis in vivo and is an important intermediate enzyme in the pathway of many pyrimidine analog drugs. In this report, we describe the isolation of the human dTMP kinase gene by functional complementation of a Saccharomyces cerevisiae cell cycle mutant, cdc8. The cDNA sequence revealed an open reading frame that encodes a protein with the molecular weight of 23,806. The deduced protein sequence was compared to known dTMP kinase sequences from different organisms. Although functionally complementary and structurally conserved, expressed human dTMP kinase in yeast shows little enzymatic activity. In contrast, active human dTMP kinase can be expressed from the gene cloned into the baculovirus expression system, as evidenced by increased enzymatic activity by four- to five-fold. Unlike yeast dTMP kinase, human dTMP kinase does not contain a cysteine residue after the conserved glycine-rich loop, but its enzymatic activity is still affected by the sulfhydryl inhibitor, 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). The levels of dTMP kinase mRNA and its enzymatic activity fluctuate during the cell cycle, peaking at the S phase. Thus, like Saccharomyces cerevisiae CDC8 (encoding dTMP kinase), the human homolog mRNA and enzymatic activity are also cell cycle regulated. We have also examined four neuroblastoma cell lines for dTMP kinase mRNA levels and its kinase activities, which appear to vary according to cell growth rate. Our results suggest that the expression of the dTMP kinase gene and its activity coincide with various stages of cell growth. The identification of the human dTMP kinase gene and expression of its product in the baculovirus expression system should facilitate study of the mechanism of gene regulation and its role in pyrimidine metabolism.