The single glycoprotein species of vesicular stomatitis virus exhibits heterogeneity in isoelectric focusing. Short term radioisotopic labeling of infected cells revealed that this heterogeneity is acquired after synthesis and glycosylation of the protein, during the period of oligosaccharide processing from the high mannose to the complex form. In this process, radioactive sulfate is incorporated randomly into different glycoprotein molecules, conferring unequal amounts of negative charge upon them. Since the virus glycoprotein is processed by cellular pathways, it is likely that differential sulfation is responsible for much of the charge heterogeneity displayed by cellular glycoproteins.