Leaf protoplasts of tobacco (Nicotlana tabacum L.) were employed for transfection of chimeric transcriptional gene fusions comprising the 35S promoter from cauliflower mosaic virus, the coding sequence of the G-protein from vesicular stomatitis virus (VSVG) and the transcriptional terminator from the Agrobacterium tumefaciens nopaline-synthetase gene. Transient expression of the chimeric gene was monitored through Northern analysis of total protoplast RNA using a labeled VSV cDNA probe, and through Western-blot analysis of protoplast proteins using a polyclonal and-VSV antiserum. Although a single species of mRNA was detected in the transfected protoplasts, two glycoproteins differing in mass by approx. 9 kDa were detected by the antiserum. Biosynthesis of the VSVG isoforms was not impeded by chemical inhibitors of cell-wall production or of proline hydroxylation. Transfection using mutant forms of the VSVG coding sequence in which either one or both consensus glycosylation sites were removed resulted in the production of progressively smaller VSVG proteins. Those proteins produced from the double mutant had mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were very similar to those produced from the wild-type construct in the presence of tunicamycin. Analysis of protoplast homogenates by differential centrifugation showed that the two VSVG isoforms were exclusively associated with cellular membranes. The larger protein co-localized with the plasma membrane and with the organelles of the endomembrane-secretory pathway leading to the plasma membrane. The smaller protein was associated with membranes of lower isopycnic densities which were not identical to the endoplasmic reticulum. The larger protein displayed greater sensitivity than did the smaller to degradation in vivo by exogenously added protease. Immunofluorescence microscopy demonstrated that the VSVG isoforms were present both within the protoplasts and at the surface of the plasma membrane. The intracellular distribution was either punctate or reticulate. These results are consistent with the progressive and accurate glycosylation of the newly synthesized VSVG polypeptide during its passage through the endomembrane-secretory pathway, the access of the larger isoform to the cell surface, and the conversion of the larger to the small isoform by selective proteolysis.