SUMMARYA homology search of proteins predicted from the recently reported complete DNA sequence of varicella-zoster virus (VZV) revealed that the product of gene 13 was highly homologous to eukaryotic and prokaryotic thymidylate synthetases (TSs). The VZV protein was shown to be a TS by three functional tests. Firstly, a plasmid designed to express the native protein was able to complement a strain of Escherichia coil in which the natural TS gene is deleted. Secondly, in an enzyme assay for TS, extracts of the complemented strain were capable of releasing tritiated water from 2'-deoxy [5-3H]uridylate. Thirdly, these extracts contained a protein that bound isotopically labelled 5-fluoro-2'-deoxyuridylate, a ligand specific for the active site of TS. In addition, a novel ligand-binding protein was detected in human cells infected with VZV.Two common human diseases, chickenpox and shingles, are caused by varicella-zoster virus (VZV). This virus is a member of the Alphaherpesvirinae, a subfamily of the Herpesviridae typified by the most extensively studied human herpesvirus, herpes simplex virus type 1 (HSV-1). Davison & Scott (1986) identified genes encoding 67 unique proteins from an analysis of the complete sequence of the 125 kbp linear double-stranded DNA genome of VZV. Five glycoprotein genes were proposed from considerations of primary amino acid sequence, and the glycoprotein products of four have been detected (Ellis et al., 1985;Davison et al., 1985;Keller et al., 1986Keller et al., , 1987. Functional assignments for 12 additional VZV proteins are dependent on comparisons with available data for HSV-1 proteins of known function (Davison & Scott, 1986). The functions of two of the remaining 50 VZV proteins may be proposed on the basis of homology with non-herpesvirus proteins. One is related to several eukaryotic protein kinases (McGeoch & Davison, 1986), and the other is homologous to prokaryotic and eukaryotic thymidylate synthetases. In this paper, we describe the structural and functional identification of the VZV thymidylate synthetase (TS) gene and the detection of a novel TS in VZV-infected cells.TS (5,10-methylenetetrahydrofolate : dUMP C-methyltransferase, EC 2.1.1.45) is a homodimeric enzyme which catalyses the reductive methylation of deoxyuridylate to thymidylate. As the sole means for supplying thymidine nucleotides de novo, TS is crucial in DNA metabolism. A particularly striking feature of this enzyme is its high degree of amino acid sequence conservation. For example, the human and Escherichia coli proteins are 53% homologous (Takeishi et al., 1985). A computer-aided homology search of the 67 VZV proteins revealed that the product of gene 13 has a similar degree of homology to published TSs. The protein-coding t Present address: