Low molecular weight heparin (FR-860), and conventional unfractionated heparin (UF-heparin) were fractionated by rabbit antithrombin III (AT III)-Sepharose, and the effects of each affinity fraction on the coagulation and fibrinolytic activities were investigated. FR-860 was fractionated to no-affinity, low-affinity (LA). and high-affinity (HA) fractions, and UF-heparin to LA and HA fractions. The HA fractions showed higher activities regarding the prolongation of activated partial thromboplastin time, anti-factor Xa activity and antithrombin activity compared with those of LA. The HA and LA fractions exhibited the enhancement of heparin cofactor II (HC II) activity and flbrinolytic activity in a dose-dependent manner. These results suggest that the antithrombotic activity of FR-860 is exerted through AT III and other mechanism such as HC II -mediated system.