A recombinant human plasminogen activator hybrid variant K2tu-PA, expressed in Chinese hamster ovary cells, is partially glycosylated at Asn12 (A chain, kringle-2 domain) and completely glycosylated at Asn247 (B chain, protease domain). After release of the N-linked carbohydrate chains by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the protein by gel permeation chromatography, then fractionated by FPLC on Mono Q, followed by HPLC on Lichrosorb-NH2, and analysed by 500-MHz 1H-NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (8%), disialylated diantennary (45%), disialylated tri- and tri'-antennary (1%), trisialylated tri- and tri'-antennary (28%), and tetrasialylated tetra-antennary (18%) structures, all having fucose in alpha(1-6)-linkage at the Asn-bound N-acetylglucosamine. Sialic acid occurred exclusively in alpha(2-3)-linkage to galactose, and consisted of N-acetylneuraminic acid (94%), N-glycolylneuraminic acid (3%), and N-acetyl-9-O-acetylneuraminic acid (3%). In addition, glycopeptide fragments corresponding with the A or B chain of K2tu-PA were analysed. The oligosaccharides attached to Asn12 are less processed than those attached to Asn247. Comparison of the glycosylation pattern of K2tu-PA with that of tissue-type plasminogen activator from different biological sources showed significant differences. Profiling studies on different K2tu-PA production batches demonstrated that the structures of N-linked oligosaccharides were identical, but that relative amounts vary with the applied isolation procedure of the chimeric glycoprotein.