The nucleoside analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) inhibited the Golgi-associated terminal glycosylation in herpes simplex virus type 1- and type 2-infected cells, specifically incorporation of galactose and sialic acid into N-linked oligosaccharides, and incorporation of sialic acid and, to a lesser extent, of galactose into O-linked oligo saccharides. This resulted in formation of viral glycoproteins with terminal GlcNAc and Fuc in N-linked oligosaccharides and terminal O-linked GalNAc. Inhibition of formation of UDP-hexoses and of acceptor glycoprotein synthesis and inhibition of cellular transport of viral glycoproteins were not observed. No evidence for the formation of a sugar nucleotide analog of BVdU was obtained. Inhibition required phosphorylation of BVdU to its 5' monophosphate (BVdUMP) by the virus-coded thymidine kinase. In a cell-free system, this monophosphate inhibited the transport of pyrimidine sugar nucleotides across Golgi membranes and, as a consequence, the incorporation of sugars into glycoproteins. Inhibition of galactosyltransferase by BVdUMP was insignificant. BVdUMP did not inhibit translocation across the Golgi membrane of purine sugar nucleotides. Inhibition of sugar nucleotide translocation represents the first target for design of virus-specific glycosylation inhibitors.