The gene encoding bovine herpesvirus 1 (BHV 1) deoxyuridine triphosphatase (dUTPase) was isolated by a PCR procedure using degenerate oligonucleotide primers whose sequences were based upon conserved motifs commonly present in dUTPase genes. This gene was found to reside between 0.059 and 0.066 map units in the BHV 1 Cooper strain. DNA sequence analysis of this region revealed an open reading frame of 975 base pairs capable of encoding 325 amino acids. The deduced amino acid sequence of the open reading frame exhibits significant homology with dUTPases of other herpesviruses (including human herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus), and it contains five conserved amino acid motifs characteristics of all dUTPases identified to date. A mutant virus carrying a partial deletion of the putative dUTPase gene was made and was found to lack virus-encoded dUTPase activity. This further confirmed that we have identified the BHV 1 dUTPase gene. In addition, a further analysis of the genomic fragment which contains the dUTPase coding sequence revealed an additional 288-base-pair open reading frame which appears to be colinear with the HSV 1 UL49.5 gene. The deduced amino acid sequence of this open reading frame is significantly homologous to the HSV 1 UL49.5 gene product, and as with UL49.5, it contains a potential signal sequence and transmembrane domain characteristic of membrane-associated proteins. These results suggest that this open reading frame represents the BHV 1 homolog of the HSV 1 UL49.5 gene. Since our dUTPase negative mutant was fully viable and since the mutant was constructed such that the UL49.5 gene was also deleted, both the dUTPase and the UL49.5 gene homolog are not required for virus growth in cell culture.