The oligosaccharide-lipid which is the precursor of asparagine-linked oligosaccharides of eucaryotic glycoproteins is synthesized from sugar nucleotides in the endoplasmic reticulum. The transmembrane location of the assembly of this oligosaccharide-lipid has been studied in vitro in rat liver microsomes. Protease treatment of these sealed vesicles which are derived from the endoplasmic reticulum resulted in the inactivation of a number of enzymes of oligosaccharide-lipid synthesis. Three early steps, the synthesis of dolichol--phosphate--mannose, of dolichol--phosphate--glucose and of dolichol--pyrophosphoryl--di--N--acetylchitobiose, as well as the final steps, the addition of glucose residues to oligosaccharide-lipid, were inactivated under conditions where only the cytoplasmic side of the membrane was accessible to protease. This finding, and the fact that no activities were latent to protease in intact microsomal vesicles, suggest that oligosaccharide-lipid is assembled on the cytoplasmic side of the microsomal membrane. However, the possibility of enzymes spanning the bilayer with their active sites facing the lumen cannot be ruled out. These results are discussed in relation to the segregation of newly made glycoprotein products within the lumen of the endoplasmic reticulum.