The sequence of the bovine herpesvirus 1 (BHV-1) gene that is homologous to the herpes simplex virus UL49 gene was determined. The BHV-1 UL49 homolog open reading frame consists of 774 bp and is capable of encoding 258 amino acids. Northern (RNA) blot analysis showed that the BHV-1 UL49 homolog is transcribed into a 1.1-kb RNA which is coterminal with the transcripts of an upstream UL49.5 homolog gene. Rabbit antisera produced against synthetic peptides of the predicted UL49 homolog gene product recognized a polypeptide of 33 to 35 kDa in both virus-infected cells and isolated virions. Further analysis by unionic-detergent partition of isolated virions suggested that the UL49 homolog gene product is a virion tegument protein. Indirect immunofluorescence assay revealed that the UL49 homolog gene product was predominantly localized in the nuclei of BHV-1-infected cells. A mutant virus with the UL49 homolog gene deleted was produced, and it was able to replicate in noncomplementing cells. Nevertheless, the yield of mutant virus was significantly reduced. The results from this study suggest that the BHV-1 UL49 homolog gene encodes a nuclear protein which constitutes a tegument component in mature virions and that it is dispensable for virus growth in cell culture.