The PLA₂ and crotapotin subunits of crotoxin from Crotalus durissus cascavella venom were purified by a combination of HPLC molecular exclusion (Protein Pack 300SW column) and reverse-phase HPLC (RP-HPLC). Tricine SDS—PAGE showed that the PLA₂ and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA₂ showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA₂ showed allosteric behavior, with maximal activity at pH 8.3 and 35–40°C. The C. d. cascavella PLA₂ required Ca²⁺ for activity, but was inhibited by Cu²⁺ and Zn²⁺ and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA₂ by acting as allosteric inhibitors.