Chicken embryo fibroblasts (C/E phenotype) infected with subgroups B and C of the Prague strain of Rous sarcoma virus were radiolabeled with either [6-(3)H]-glucosamine or [2-(3)H]mannose, and virus was purified from the growth medium. The large envelope glycoprotein, gp85, was the only major radiolabeled component of purified virus. Pronase-digested glycopeptides from purified virus were analyzed by a combination of (i) gel filtration with columns of Sephadex G15/G50 and Bio-Gel P4 and (ii) enzymatic digestion of the oligosaccharide chains with specific exoglycosidases and endo-beta-N-acetylglucosaminidases. The rather broad molecular weight distribution (approximately 2,000 to 4,000) for glycopeptides in these studies and previous studies in other laboratories was shown to represent actual heterogeneity in the carbohydrate moieties: (i) the glycopeptides contained both mannose-rich, neutral chains and complex, acidic chains with terminal sialic acid; and (ii) both classes of asparagine-linked carbohydrate structures exhibited heterogeneity in the size of the oligomannosyl core (a mixture of approximately 5 to 9 mannose units for the neutral structures, and 3 or 5 mannose units for the acidic structures). With the [2-(3)H]mannose-labeled glycopeptides from Rous sarcoma virus, Prague strain subgroup C, most of the oligosaccharide chains were high-molecular-weight, acidic structures, with similar numbers of 3-mannose and 5-mannose core structures.