Abstract The purified anticoagulant principle AAV-XaI isolated from the snake venom of Agkistrodon acutus binds to purified bovine Factor Xa in the presence of calcium ions. The complex has amidolytic activity with S-2222. AAV-XaI inhibits the conversion of prothrombin to thrombin by the prothrombinase complex, but has no influence on the amidolytic activity of Factor Xa. AAV-XaI binds to Factor X more effectively than to Factor Xa. The complex of Factor X ·Ca 2 +· AAV-XaI yields only amidolytic activity after activation with RVV-X. In 25% sodium citrate solution AAV-XaI does not modify the generation of thrombin from prethrombin 1 by Factor Xa. β-Factor Xa-E is a form of Factor Xa with only amidolytic activity and is not inhibited or bound by AAV-XaI. The venom inhibitor covers binding sites on the light chain of Factor Xa that are necessary for phospholipid function.