Bothrops lanceolatus venom contains a variety of enzymatic and biological activities. The present work investigated the hemolytic activity of this venom and its phospholipase A 2 (PLA 2 ). Bothrops lanceolatus venom (6.7 µg/mL) caused indirect hemolysis of cow, horse, rat and sheep erythrocytes, with horse erythrocytes being the most sensitive; no direct hemolysis was observed. Hemolysis in sheep erythrocytes was concentration-dependent (5-11.7 µg/mL) and markedly attenuated by heating the venom for 30 minutes at ≥ 40°C and by the PLA 2 inhibitor p-bromophenacyl bromide. An acidic PLA 2 (5 µg/mL) purified from B. lanceolatus venom also caused hemolysis. This PLA 2 showed immunoprecipitin lines with antivenom against B. lanceolatus, which suggests that the enzymatic and hemolytic activities of this enzyme may be neutralized during antivenom therapy. These results indicate that B. lanceolatus venom and its PLA 2 can cause hemolysis in vitro.