Feline herpesvirus 1 (FHV) is the causative agent of viral rhinotracheitis in cats. Current vaccination programs employing attenuated live and killed FHV vaccines have been effective in reducing the incidence of this disease. As an initial step in the development of recombinant FHVs for use in the vaccination of cats, we have identified the thymidine kinase (TK) gene of this feline-specific alphaherpesvirus. Comparisons of the amino acid sequences of other herpesvirus TK proteins have shown that these proteins are highly divergent, sharing only short regions of imperfect amino acid identity. We have used the polymerase chain reaction method of DNA amplification to increase the specificity associated with the use of short, highly degenerate oligonucleotide probes derived from regions of imperfect amino acid conservation. These methods were used to isolate the TK gene of FHV and should prove to be useful in the identification of new members of other viral and cellular gene families. A recombinant FHV bearing a deletion in the identified TK gene was constructed and shown to possess the expected TK- phenotype. The FHV TK gene is located at a position of approximately 40% in the long unique component of the FHV genome. The location of the TK gene and the location and orientation of flanking FHV genes, homologs of herpes simplex virus type 1 UL24 and UL22, are conserved among alphaherpesviruses.