Snake venom metalloproteinases (SVMPs) have been extensively analyzed and have been shown to be active on plasma and extracellular matrix proteins and therefore they are involved in the hemostasis imbalance observed upon envenomation. Structurally they are classified as the following: P-I (SVMPs having only a metalloproteinase domain); P-II (SVMPs being synthesized with metalloproteinase and disintegrin domains); P-III (SVMPs being synthesized with metalloproteinase, disintegrin-like and cysteine-rich domains) and P-IV (SVMPs having the P-III domain structure plus two lectin-like domains connected by disulfide bonds). HF3 is a P-III class enzyme from the venom of Bothrops jararaca that shows high hemorrhagic activity and low caseinolytic activity. BJ-P1 was isolated from the venom of B. jararaca in this work by a one-step procedure using gel filtration chromatography in FPLC system. Based on the molecular mass of BJ-P1 obtained by SDS-polyacrylamide gel electrophoresis and on the identification of the protein by mass spectrometry as a SVMP, it was possible to classify BJ-P1 as a P-I class enzyme. This enzyme is highly active on casein, however it is not able to cause hemorrhage. Both HF3 and BJ-P1 degraded various plasma proteins, such as fibrinogen, fibrin, fibronectin and vitronectin, as well as the extracellular matrix proteins collagen type IV, collagen type VI and laminin showing different hydrolysis profiles. The preliminary results of this study suggest that the presence of disintegrin-like and cysteine-rich domains in HF3 confer to this enzyme substrate specificity different than that of BJ-P1, which is important for the in vivo hemorrhagic activity displayed by HF3.