The fibrinolytic enzyme of 51,000 Da isolated from the venom of Agkistrodon halys lyses fibrinogen and fibrin polymer in the absence of plasminogen without having thrombinlike activity. Treatment of the enzyme with diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride resulted in complete loss of the fibrinolytic activity, suggesting that the enzyme is a member of serine protease family. Proteolytic activity of the enzyme is also inhibited by incubation with metal ions such as $Zn^{2+}$, $Cu^{2+}$, $Cd^{2+}$, $Li^{2+}$, and $Co^{2+}$. The enzyme is relatively stable at room temperature and its maximal catalytic activity is attained at pH 8.0, $37^{circ}C$. The ${alpha}$ chain of fibrinogen or fibrin polymer is more sensitive to the proteolytic degradation by the enzyme than the ${eta}$ chain, while the ${gamma}$ chain is not affected at all. Hydrolysis of the synthetic peptide substrates including N-benzoyl-Phe-Val-Arg-p-nitroanilide, which is a specific substrate for angiotensin I-converting enzyme, suggests the venom fibrinolytic enzyme cleaves after arginine residue. The enzyme was not able to hydrolyze human serum albumin, plasminogen, thrombin, immunoglobulin, and tissue-type plasminogen activator. Since the enzyme is active in human plasma and the degree of fibrinolysis was proportional to the amount of enzyme added, it is postulated that the enzyme can mediate fibrinolytic function in vivo.