O-Linked oligosaccharide chains were identified in the activation peptide (AP) of human blood coagulation factor IX. The peptide obtained from human factor IX was separated into three molecular species (AP alpha, AP beta, and AP gamma) by reversed-phase high-performance liquid chromatography. Amino acid analysis showed that AP alpha, but not AP beta and AP gamma, contained galactosamine in addition to glucosamine, thereby suggesting the presence of an O-linked sugar chain(s) in the molecule of AP alpha. A nonapeptide (AP alpha-D4, residues 157-165) and an undecapeptide (AP alpha-D5, 166-176) derived from AP alpha contained Thr-159 and Thr-169, neither of which could be identified using a gas-phase protein sequencer. All other serine and threonine residues present in AP alpha were identified by peptide sequencing. Component sugar and sialic acid analyses of AP alpha-D4 and AP alpha-D5 revealed that they contained 1 mol each of N-acetyl-D-galactosamine (GalNAc), D-galactose (Gal), and sialic acid. Fast atom bombardment tandem mass spectrometric analysis of AP alpha-D4 suggested the existence of Gal-GalNAc-Thr, NeuNAc-(Gal-)GalNAc-Thr, and NeuNAc-Gal-GalNAc-Thr structures. On the basis of amino acid analysis after the isolation of AP alpha, it accounted for approximately 35% of the total activation peptide obtained. From these results, it was concluded that a part of the activation peptide of human factor IX in circulating blood has tri- and tetrasaccharides O-glycosidically linked to the threonine residues at 159 and 169.