Benzhydrazone is a bis-amidinohydrazone derivative which specifically hinders the formation of herpes simplex virus (HSV) glycoproteins. In this study we present some structural features of the oligosaccharide chains of herpesvirus glycoproteins synthesized in cells incubated with the inhibitor. Gel filtration analysis of glycopeptides, obtained through exhaustive pronase-digestion of infected cells after a long or a short labeling with 14C-glucosamine, showed that benzhydrazone reduced the appearance of glycopeptides of all the size-classes, including the mannose-rich glycopeptide with an approximate molecular weight of 1500. The same percent of label was released from both untreated and benzhydrazone-treated cells after digestion with endo-beta-N-acetylglucosaminidase H, an enzyme which cleaves between the N-acetylglucosamine residues in large high-mannose type oligosaccharides. This indicates that the relative amount of glycoproteins sensitive to this enzyme did not differ in the two kinds of samples. PAGE analysis confirmed that the same glycoproteins were digested in both samples. They were gA, pgC, and pgD, which therefore contain high-mannose type oligosaccharides. It is concluded that benzhydrazone hinders carbohydrate addition to herpesvirus proteins at an early step.