Phospholipases A2 (PLsA2) are abundant in the snake venoms and are able to interfere on the course of several biological processes including thrombosis by affecting blood coagulation and platelet aggregation. They catalyze the hydrolysis of the sn-2 ester bond of 1,2-diacyl-3-phosphoglycerides to produce lysophosphatidylcholine and fatty acid. The Ca²⁺ bound to the putative calcium-binding loop seems to be essential for PLsA2 activity. As little is known about the toxins of Bothrops leucurus venom, this work was developed to purify proteins with phospholipase A2 (PLA2) activity from Bothrops leucurus snake venom. Two PLsA2 were purified to homogeneity through three chromatographic steps: conventional gel filtration on Sephacryl S200, ion-exchange on Q-Sepharose FPLC system and reverse phase HPLC on Vydac C4. The molecular mass for both enzymes was estimated to be 14 kDa by SDS-PAGE.  The sequences of the first forty-eight amino acids at the N-terminal sequence of each protein were determined by automated Edman degradation and are LFELGKMILQETGKNSVKSYGVYGCNCGVGGRGKPKDATDKCCYVHK and DLWQFGQMILKETGKLPFPYYTTYGCYCGWGGQGQPKDATDRCCFVHD. These sequences show that one enzyme presents lysine at position 48 (Lys48) and the other an aspartic acid in this position (Asp48), and therefore they were designated blK-PLA2 and blD-PLA2 respectively. PLA2 activity was determined fluorometrically using phospholipids labeled at the sn-2-acyl position with 10-pyrenyldecanoic acid and the specific activity was expressed as activity units/mg of protein. One activity unit was defined as 1 nanomol of the fluorescent product (10-pyrenyldecanoic acid) liberated/min. Protein concentration of samples was estimated by the Coomassie Blue method. The specific phospholipase A2 activity was 25.0 and 433.9 activity units/mg of protein for blK-PLA2 and blD-PLA2, respectively. No PLA2 activity was observed in the absence of CaCl₂ for both enzymes.