Anophelin is a 6.3 kDa peptide previously characterized from the salivary gland of Anopheles albimanus (Valenzuela et al., Biochemistry 1999; 38, 11209-15). It is a potent and specific thrombin inhibitor that interacts with both the catalytic site and the anion-binding exosite 1 of the enzyme. In this work we show that Anophelin is an effective anticoagulant in vitro, as determined by PT and APTT coagulation tests using citrated rat plasma. We have also investigated the effects of Anophelin in vivo, using a venous thrombosis model. Intravenous administration of the drug in rats caused a dose-dependent reduction in thrombus formation. We observed that Anophelin doses of 250 mg/kg body weight inhibited ~55% of thrombus formation, while complete inhibition was attained at 500 mg/kg. Further evaluation of the bleeding effect is necessary to determine the occurrence of side-effects. In conclusion our results demonstrate that Anophelin is an effective antithrombotic agent in vivo.