The effect of molecular weight and sulfate amount of sulfated polysaccharide on the thrombin inhibitory activity of heparin cofactor II was investigated by using various dextran sulfate fractions with different molecular weight and sulfur content. The activity of dextran sulfate fractions of each size increased as the sulfur content was increased from 9 to 18%, and the activity decreased in molecules below 10 kDa. The maximum second order rate constant of heparin cofactor II-thrombin reaction in the presence of the fractions of over-10 kDa and 18% sulfur was 2.7 X 10(8) M-1 min-1 that was almost same as in the presence of heparin or dermatan sulfate. On the other hand, dextran sulfate accelerated antithrombin III-thrombin reaction only about 40-fold less than heparin. These results indicate that a large molecular size and significant amount of sulfate groups are only essential in the acceleration of the thrombin inhibitory activity of heparin cofactor II, whereas a specific sequence of heparin is required to that of antithrombin III.