Biosynthesis of N-glycosylated glycoproteins in eukaryotes. N-glycosylation is initiated at the endoplasmic reticulum (ER) membrane using nucleotide sugar donor substrates and a membrane-bound acceptor phospholipid with multiple isoprenyl units (dolichol-phosphate, P-Dol). The first sugar (GlcNAc) is transferred as GlcNAc-phosphate from UDP-GlcNAc by GlcNAc-P-transferase, resulting in GlcNAc-diphosphate-dolichol (GlcNAc-PP-Dol). This step can be inhibited by the UDP-GlcNAc analog tunicamycin. On the outside face of the ER membrane, another GlcNAc is added to form chitobiose, followed by five Man residues to form a heptasaccharide (Man5GlcNAc2)-PP-Dol. This heptasaccharide is flipped to the inside of the ER where the chain grows by transfer of sugars from membrane-bound Man-P-Dol and Glc-P-Dol. The completed saccharide Glc3Man9GlcNAc2 is then transferred by an oligosaccharyltransferase complex (OST) to the Asn residue in an Asn-x-Ser/Thr sequon of nascent proteins. After trimming of sugar residues in the ER by removal of Glc and Man residues to the Man8GlcNAc2 structure, glycoproteins are exported to the Golgi where further trimming occurs by mannosidases. Many N-glycan chains are processed to the complex type by the addition of GlcNAc residues by GlcNAc-transferases I to V (MGAT1 to 5). Chains grow further by the addition of Gal-GlcNAc sequences and termination by sialyl-, Fuc-, Gal-, GlcNAc-, and GalNAc-transferases, which are all highly specific for both the donor and the acceptor substrates and with few exceptions form only one type of linkage between sugars. This creates a multitude of hundreds of different structures and epitopes with many possible functions, depending on the final destination of the glycoprotein, e.g., in the cell membrane or in secretions. Glycoprotein biosynthesis is regulated at many different levels, e.g., by the synthesis and delivery of nucleotide sugar substrates, the expression, activities and localization of glycosyltransferases and trimming hydrolases, the competition of enzymes for common substrates, levels of metal ion activating factors, localization of enzymes involved, and rate of transport of glycoproteins.