The thymidine kinases from feline herpesvirus (FHV TK) and canine herpesvirus (CHV TK) were cloned and characterized. The two proteins are closely sequence-related to each other and also to the herpes simplex virus type 1 thymidine kinase (HSV-1 TK). Although FHV TK and CHV TK have a level of identity of 31 and 35%, respectively, with HSV-1 TK, and a general amino acid similarity of approximately 54% with HSV-1 TK, they do not recognize the same broad range of substrates as HSV-1 TK does. Instead the substrate recognition is restricted to dThd and pyrimidine analogs such as 1-beta-d-arabinofuranosylthymine (araT), 3'-azido-2',3'-dideoxythymidine (AZT) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). FHV TK and CHV TK differ in substrate recognition from mammalian cytosolic thymidine kinase 1 (TK1) in that TK1 does not phosphorylate BVDU and they also differ from mammalian mitochondrial thymidine kinase 2 (TK2), which, in addition to thymidine and thymidine analogs also phosphorylates dCyd. Although the nucleoside analog BVDU was a good substrate for FHV and CHV TK, the compound was poorly inhibitory to virus-induced cytopathic effect in FHV- and CHV-infected cells. The reason is likely the poor, if any, thymidylate kinase activity of FHV and CHV TK, which in HSV-1 TK-expressing cells convert BVDU-MP to its 5'-diphosphate derivative.