The role of glycosylation in the biosynthesis, processing, and shedding of the murine mammary tumor virus (MuMTV) glycoproteins and in virus production was investigated in a clonal mammary tumor cell line, GR-3A, using two inhibitors of protein glycosylation, 2-deoxyglucose (2-DG) and tunicamycin (TM). It was found that both 2-DG and TM completely inhibited the synthesis of the MuMTV envelope precursor polyprotein, Pr70env, and, as a consequence, the synthesis of the viral glycoproteins gp52 and gp36. By contrast, the synthesis of Pr73gag, the polyprotein precursor of the internal structural proteins of the virus, was only inhibited by 10-15% by 2-DG and TM. Although 2-DG and TM blocked the synthesis of Pr70env, a new polypeptide, related to gp52 and gp36, with a mol wt of 60,000 (P60env) was found to be synthesized in the treated cells. The P60env molecules appeared to be degraded intracellularly since they were not found to (1) undergo site-specific cleavage; (2) accumulate inside the cell or on the cell surface; (3) be secreted into the culture medium; and (4) be incorporated into the virions produced during the drug treatment. In spite of the lack of gp52 and gp36 synthesis in the presence of TM and 2-DG, mature MuMTV particles containing the characteristic surface projections known to be composed of gp52 and gp36 continued to be assembled and released at a reduced rate for at least 30 hr. In addition, the buoyant density and the polypeptide composition of the particles were found to be identical to virions produced by untreated cells. Thus, the virions assembled and released during 2-DG and TM treatment were not defective. Our investigations into the origin of gp52 and gp36 in these particles revealed that both molecules were synthesized prior to 2-DG and TM treatment and continued to be incorporated, along with the newly synthesized viral core proteins, into budding virions during the drug treatment. Furthermore, we found that gp52 and P75env (an aberrant form of Pr70env) that were not incorporated into virions continued to be shed normally from the cell during drug treatment. In conclusion, our results suggest that MuMTV assembly is not dependent on the synchronized synthesis of the viral core and envelope polypeptides, and that the assembled virions contain the correct ratio of these polypeptides, even when their ratio in the cell varies.