A major anticoagulant fucoidan (F-19) was purified from a commercial fucoidan fraction, which was obtained from the brown seaweed Fucus vesiculosus, by repeated gel filtration and anion-exchange chromatography. Two oversulfated fucoidans (sulfate content, 46.2% and 55.0%, respectively) were prepared from F-19 (sulfate content, 34.0%) by chemical sulfation. The antithrombin effects of the fucoidans were examined for their abilities to inhibit amidolysis of thrombin for a synthetic substrate in the presence of heparin cofactor II (HC II) and firbinogen clotting by thrombin in the absence of the serine protease inhibitor. The inhibitory effects of the oversulfated fucoidans in both assays were greater than those of the parent fucoidan with increase in the degree of sulfation, indicating that both the direct and the HC II-mediated antithrombin activities of alpha(1-->2)-fucoidan are dependent on its sulfate content.