Cytosol thymidine kinase (TK) activity is enhanced at 6 hr after bovine embryo tracheal (EBTr) and rabbit skin fibroblast (RAB-9) cells are infected with the Los Angeles and Cooper strains of bovine herpesvirus type 1 (BHV-1). To learn whether this enhancement resulted from the induction of a virus-specific TK activity, biochemical and genetic studies were carried out. The biochemical experiments demonstrated that: (i) the BHV-1-induced TK activity had a relative disc PAGE mobility (Rm) characteristic of other herpesvirus-encoded TKs and distinctly different from the Rm value of the cytosol TK of host cells; and (ii) the BHV-1-induced TK was significantly more sensitive to competitive inhibition by arabinosylthymine (araT) than the cytosol TKs of EBTr and RAB-9 cells. The genetic experiments entailed the isolation of a bromodeoxyuridine (BrdUrd)-resistant rabbit cell line [RAB(BU)] deficient in cytosol TK activity and of BrdUrd- and araT-resistant BHV-1 mutants. RAB(BU) cells acquired TK activity after they were infected by wild-type, TK+ BHV-1, but not drug-resistant BHV-1 mutants. The experiments strongly suggest that wild-type BHV-1 induces a virus-specific TK activity.