A hemorrhagic metalloproteinase, named BaH4, was isolated from the venom of the snake Bothrops asper by a combination of ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sephacryl S-200. BaH4 is a 69 kDa protein with a pI of 5.3. It was recognized by antibodies raised against hemorrhagic metalloproteinase BaH1 isolated from B. asper venom, with a reaction of partial immunologic identity. BaH4 shows proteolytic activity on biotinylated casein, hide powder azure and fibrin, although having lower activity than crude B. asper venom and metalloproteinase BaP1 isolated from the same venom. BaH4 hydrolyzed fibronectin, laminin and type IV collagen in vitro, albeit at a relatively high enzyme:substrate ratio. Proteolytic activity was inhibited by chelating agents and 2-mercaptoethanol, but not by soybean trypsin inhibitor. Prominent hemorrhage developed in gastrocnemius and cremaster muscles after administration of BaH4. Moreover, it induced lethality in mice after intravenous injection, with an LD50 of 0.37 microg/g. Histological observations showed conspicuous pulmonary hemorrhage when the enzyme was injected intravenously. BaH4 is a hemorrhagic metalloproteinase which may play a relevant role in local and systemic bleeding characteristic of B. asper envenomations.