The effect of some sulfated derivatives of chitosan on several blood-coagulant factors was examined in comparison with those of heparin (174 units/mg). The anticoagulant activity (units/mg) with respect to activated partial thromboplastin time was 331-379 for O-sulfated N-acetylchitosan (2), 190-287 for N, O-sulfated chitosan (1), and 21-31 for sulfated O-carboxymethylchitosan (3). The activity (units/mg) with respect to thrombin time was 70-87 for 2, 44-70 for 1, and 14-22 for 3. The activity (units/mg) with respect to antithrombin activity was 4.9-9.0 for 2, 4.7-8.7 for 1, and 3.3-6.0 for 3. No anti-(factor Xa) activity was observed with 1-3. A 6-sulfate group in the hexosaminyl moiety was a main active site; although a 3-sulfate group was not essential, it promoted the activity of the 6-sulfate group. N-Sulfate was not a prerequisite for activity. The biological activities were also related to molecular weight in the sequence 2 (26,000) greater than heparin (21,000) greater than 1 (12,000) greater than 3 (540,000). For the Methylene Blue complexes, 1, but neither 2 nor 3, exhibited a negative, induced Cotton effect similar to that of heparin.