(E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVdU) was phosphorylated by the bovine herpesvirus 1 (BHV-1)-induced thymidine kinase and subsequently incorporated into viral DNA, resulting in DNA that was more dense than DNA from untreated cells. Incorporation of the drug did not result in the termination of replicating BHV-1 DNA molecules since radioactively labeled DNA synthesized in drug-treated and untreated cells sedimented at similar rates in alkaline sucrose gradients. No differences were observed in the electrophoretic mobility of [35S]methionine-labeled viral polypeptides synthesized in treated and untreated cells, although [3H]glucosamine-labeled viral glycoproteins synthesized in treated cells were of a lower molecular weight than those in untreated cells. In BVdU-treated cells, unlike untreated cells, immature neutral and basic precursors of the mature viral glycoproteins accumulated. Although BVdU-treated and untreated cells contained similar amounts of virus, very little virus was released into the culture supernatant from BVdU-treated cells. Our results suggest that BVdU partially inhibits the glycosylation of BHV-1 glycoproteins. BVdU-sensitive glycosylation, however, is not necessary for expression of these glycoproteins on the surface of infected cells since the glycoproteins could be labeled on intact cells with 125I and because BVdU-treated cells remained sensitive to antibody-dependent, cell-mediated cytotoxity mediated by anti-BHV-1 serum. The phosphorylation of BVdU was a prerequisite for its effect on glycosylation since the glycoproteins of a thymidine kinase-deficient mutant of BHV-1 were not affected.