The transmembrane orientation of Glc3Man9GlcNAc2-pyrophosphoryl-dolichol, which is the oligosaccharide donor in the glycosylation of asparagine residues of eukaryotic glycoproteins has been investigated. The lectin concanavalin A was used as a nonpenetrating probe to study the location of this oligosaccharide-lipid in microsomal vesicles prepared from cultured fibroblasts. Lectin treatment of intact vesicles, and vesicles made leaky with low concentrations of detergent showed that this oligosaccharide-lipid is on the luminal side of membrane. The oligosaccharide-lipid was bound by lectin only if the permeability barrier of the membrane had been destroyed by detergent; very little binding was seen in intact vesicles. This result suggests that glycosylation of nascent secretory and membrane glycoproteins occurs on the luminal side of the membrane. It also implies that sugar residues derived from cytoplasmic sugar nucleotides must be transported across the membrane at some point during the synthesis and accumulation of mature, luminal oligosaccharide-lipid, although the identity of the transported species remains unknown.