Human tissue factor pathway inhibitor is a protease inhibitor with three tandem Kunitz-type inhibitory domains. The recombinant protein (r-hTFPI) was produced using Chinese hamster ovary cells, and its polypeptide and carbohydrate chain structures were analyzed. The complete amino acid sequence, composed of 276 residues, was determined using a protein sequencer after protease digestion and it was identical to that predicted from the cDNA sequence. Among three potential N-glycosylation sites, both Asn¹¹⁷ and Asn¹⁶⁷ were fully N-glycosylated but Asn²²⁸ was not. Thr¹⁷⁵ was also fully O-glycosylated, but Ser¹⁷⁴ was partially O-glycosylated. Carbohydrate composition and mass spectrometric analyses of the undecapeptide OG-11 (residues Leu¹⁷⁰∼Leu¹⁸⁰) showed that two O-linked carbohydrate chains consisted of a type-1 core structure (Gal-GalNAc-Ser/Thr) with 0−3 mol of N-acetylneuraminic acid(s). The N-linked carbohydrate chains were analyzed by two-dimensional carbohydrate mapping combined with sequential glycosidase digestion, after the reducing-ends of carbohydrate residues were tagged with 2-aminopyridine and non-reducing-end sialic acids were removed with sialidase. All the N-linked structures in r-hTFPI were complex-type carbohydrate chains with one fucose residue attached to the reducing-end GlcNAc and consisted of bi-, tri-, and tetraantennary carbohydrate chains in the ratio 1.9:1.3:1.0. Fucosylated tri- and tetraantennary carbohydrate chains with one or two N-acetyllactosaminyl repeats were also found (30% of carbohydrate chains determined). Thus, the region between Kunitz domains 2 and 3 encoded by exon 7 was highly glycosylated by two O-linked carbohydrate chains at Ser¹⁷⁴ and Thr¹⁷⁵ and one N-linked carbohydrate chain at Asn¹⁶⁷. These results indicated that the region is occupied by a cluster of three bulky and acidic carbohydrate chains.