Glycoproteins D1 (gD1) and D2 (gD2) of herpes simplex virus type 1 and type 2, respectively, were purified from infected HEp-2 cells labelled with [3H]glucosamine for 14 h followed by a 3 h chase using HD1 monoclonal antibody linked to Sepharose. O-linked oligosaccharides were found to be present in both glycoproteins. The identification of N-acetyl [3H]galactosaminitol as the major labelled component in the oligosaccharides generated by mild alkaline borohydride treatment demonstrated that these chains have N-acetylgalactosamine at the reducing end. These oligosaccharides consist of mono- and disialylated species with a predominance of the latter species in gD1. Size analysis and radioactive amino sugar composition strongly suggest a structure in which the galactosyl-N-acetylgalactosamine core is substituted with one or two sialic acid residues. In terms of [3H]glucosamine-derived radioactivity, O-linked oligosaccharides are less represented than N-linked oligosaccharides. The O-linked oligosaccharide number determination showed that gD1 and gD2 carry two and three chains, respectively.