The assembly of N-linked glycoproteins in eukaryotic cells begins with the segregation of these molecules within the lumen of intracellular vesicles. Since the sugar nucleotides are cytoplasmic molecules, translocation of the sugar moiety across the membrane appears as a crucial event in the glycoprotein synthesis. This N-glycosylation process occurs in two different cytological sites: in the rough endoplasmic reticulum, the stepwise synthesis of a large lipid-linked oligosaccharide takes place, as well as its transfer to protein; then after trimming the immature glycoprotein is further elongated in the Golgi apparatus. In this paper, a brief review will be given of the present knowledge on the sugar donor transport across the membrane barrier to the glycosylation site. Based upon the transmembrane orientation of oligosaccharide lipid intermediates and on the localization of the glycosyltransferase active sites, the different processes required to translocate the sugar moieties during the preassembly of the dolichyl-pyrophosphate-oligosaccharides will be examined. Combining the different results, obtained in several laboratories, it is suggested that the Man5-GlcNAc2-lipid is synthesized on the cytoplasmic side directly from the sugar-nucleotides and then translocated to the lumenal face where the Glc3-Man9-GlcNAc2-lipid is completed using Man-P-Dol and Glc-P-Dol as transmembrane carriers of these sugars. Concerning the elongation process leading to assembly of the antennae of N-acetyllactosamine type oligosaccharides, specific carriers for sugar nucleotides have been described as Golgi markers. Several authors have characterized such carriers for UDP-Gal, GDP-Fuc, CMP-NeuAc, UDP-GlcNAc and UDP-Glc using microsomal vesicles and similar results have been obtained in our laboratory using plasma membrane permeabilized cells. This carrier-mediated process leads to the formation of an intralumenal pool whose biological significance will be discussed. The translocation process of sugar donors occurring in the rough endoplasmic reticulum via lipid intermediates as well as in the Golgi apparatus via specific carriers would represent a regulation step based on the availability of the substrates for the glycosylation.