Herpesvirus saimiri (HVS) is the prototype member of a distinctive subset of lymphotropic herpesviruses (the gamma 2 subgroup) with A+T-rich coding sequences. In this paper, we show that cells productively infected with HVS contain high concentrations of a virus-specified thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45); we identify the active polypeptide and present the sequence of the virus gene. The predicted amino acid sequence of the 294-residue subunit of the virus enzyme is 70% homologous with the sequence of the human enzyme and about 50% homologous with prokaryotic thymidylate synthases, illustrating the remarkable structural constraints imposed by the thymidylate synthase function. However, the presence of the enzyme is not a conserved property of herpesviruses. We find no evidence for a virus-encoded thymidylate synthase activity (or a homology to a thymidylate synthase sequence) in G+C-rich representatives of alpha 1 (e.g., herpes simplex viruses, 66-68% G+C), beta (i.e., human cytomegalovirus, 58-59% G+C), and gamma 1 (i.e., Epstein-Barr virus, 60% G+C) herpesvirus subgroups. The production of excess thymidylate by a virus thymidylate synthase in cells infected with an A+T-rich herpesvirus would provide one plausible source of biased mutations by the virus-encoded replicative enzymes, which we have previously suggested as the likely general cause of differences in the mean nucleotide compositions of herpesvirus genomes.