1. Agkistin, purified from the snake venom of Formosan Agkistrodon acutus, belongs to the family of C-type lectin GPIb binding proteins. It is a heterodimeric molecule, consisting of alpha- (16.5 kDa) and beta- (15.5 kDa) subunits with a molecular mass of 32,512 Daltons examined by SDS - PAGE and mass spectrometry. 2. In vitro, agkistin concentration-dependently inhibited ristocetin-induced human platelet agglutination and aggregation in the presence of vWF. It also inhibited TXA2 formation and prolonged the latent period in triggering aggregation by a low concentration of thrombin (0.03 u x ml(-1)). 3. 125I-agkistin specifically bound to unactivated human platelets in a saturable manner with a KD value of 223+/-10.6 nM. This binding reaction was rapid and reversible. Monoclonal antibodies, AP1 and 6D1 raised against platelet GPIb, almost completely blocked 125I-agkistin binding to platelets. However, monoclonal antibody 7E3 raised against GPIIb/IIIa complex, trigramin, a GPIIb/IIIa antagonist, ADP and EDTA did not affect 125I-agkistin binding reaction. 4. Agkistin (250 microg x kg(-1)) significantly prolonged the bleeding time and induced transient thrombocytopenia of mice when given intravenously. Furthermore, it markedly inhibited platelet plug formation in irradiated mesenteric venules of fluorescein-treated mice in vivo. 5. In conclusion, agkistin inhibits ristocetin induced platelet aggregation mainly through its specific binding to platelet GPIb, thereby blocking the interaction between GPIb and vWF. In addition, agkistin exhibits antithrombotic activity in vivo.