Two major crotamine isoforms (III-4 and III-7) were obtained combining two chromatographic steps on molecular exclusion chromatography (Sephadex G-75) and ion-exchange column (Protein Pack SP 5PW) of the rattlesnake Crotalus durissus cumanensis venom. The " in vivo" myotoxic effect of the venom, its " in vitro" cytotoxicity in myoblasts and myotubes (C2C12) and the neurotoxic and edema-forming activity were characterized. The molecular masses of the crotamine isoforms were 4907.94 Da (III-4) and 4985.02 Da (III-7) and, as determined by mass spectrometry, both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing through tandem mass spectrometry was used to determine the primary structure of both isoforms. III-4 and III-7 isoforms presented a 42-amino acid residues sequence and showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. In vivo, both crotamine isoforms induced myotoxicty and a systemic interleukin-6 response upon intramuscular injection. These new crotamine isoforms induced low cytotoxicity in skeletal muscle myoblasts and myotubes (C2C12) and both induced a facilitatory effect on neuromuscular transmission in young chick biventer cervicis preparation. Edema-forming activity was also analyzed by injection of the crotamine isoforms into the right paw, since both crotamine isoforms exert a strong pro-inflammatory effect. © 2010 Elsevier Ltd.