The primary structure of the major N-linked carbohydrate chains attached to Asn302 of urinary-type plasminogen activator (urokinase) have been determined. Urokinase was completely deglycosylated with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F from Flavobacterium meningosepticum. Released oligosaccharides were separated from the remaining protein using gel-permeation chromatography on Bio-Gel P-100, and then on Bio-Gel P-6. Fractionation of the oligosaccharides was achieved by a combination of FPLC anion-exchange chromatography on Mono Q HR 5/5 and amine-adsorption HPLC on LiChrospher 100-NH2. Analysis by 1H-NMR spectroscopy demonstrated that the collection of N-glycans comprises di-, tri-, and tri'-antennary structures. The glycans contain predominantly GalNAc beta 1-4GlcNAc beta instead of Gal beta 1-4GlcNAc beta elements. The GalNAc residue is mainly sulfated at O4, or to a lesser extent it bears N-acetylneuraminic acid at O6; alternatively the GlcNAc residue can be fucosylated at O3. The major component, which accounts for more than 30 mol/100 mol of the total oligosaccharide pool, consists of an (alpha 1-6)-fucosylated diantennary N-linked carbohydrate chain with (SO4-)-4GalNAc beta 1-4GlcNAc beta 1-2 antennae.