Kinetics of Tupaia herpesvirus strain 2 (THV-2) DNA synthesis was measured by labeling with radioactive thymidine and determining the amount of newly synthesized DNA. Viral DNA synthesis reached a maximum between 24 to 36 hr postinfection, preceded by a transient stimulation of host cell DNA synthesis. Blot hybridizations of labeled recombinant plasmids harboring inserts of both genomic termini of THV-2 and of individual viral terminal DNA fragments to DNA of virus-infected cells and combined restriction enzyme analysis of labeled intracellular viral DNA revealed the presence of linear concatemeric and/or circular viral DNA molecules.