The O-linked oligosaccharides on mature forms of herpes simplex virus type 1 (HSVl) glycoproteins were characterized, and were found to account largely for the lower electrophoretic mobilities of these forms relative to the mobilities of immature forms. Other posttranslational modifications of HSVl glycoproteins(designated gB, gC, gD and gE) were related temporally to the discrete shifts in electrophoretic mobilities that signal acquisition of the O-linked oligosaccharides. Fatty acid acylation (principally of gE) could be detected just prior to the shifts, whereas conversion of high-mannosetype N-linked oligosaccharides to the complex type occurred coincident with the shifts. The addition of O-linked oligosaccharides did not occur in cells treated with the ionophore monensin or in a ricinresistant cell line defective in the processing of N-linked oligosaccharides. We conclude that extension of O-linked oligosaccharide chains on HSVI glycoproteins, and probably also attachment of the first O-linked sugars, occurs as a late posttranslational modification in the Golgi apparatus.