Ammodytin L (AMDL) is a myotoxic phospholipase-like protein from the venom of Vipera ammodytes with a serine in position 49 instead of an aspartate, therefore this toxin is devoid of phospholipase activity, and the membrane-damaging effect does not involve any step of phospholipase activity. The aim of the present study was to analyze the effect of AMDL on L-6 cells from rat skeletal muscle to investigate its mechanism of action and the role of calcium ions in its muscle-damaging activity. Our data indicate that the effect of ammodytin L is strongly dependent on the degree of cell differentiation. Low doses of myotoxin gave rise to a marked release of creatine kinase in myotubes differentiated from L-6 myoblasts and the presence of calcium ions plays a role in the cytotoxic effect. The presence of EGTA in the incubation buffer reduced by 50% the release of creatine kinase. No membrane damage was observed in myoblasts, but there was a significant increase of intracellular calcium concentration measured with Fura-2. A non-specific membrane effect of AMDL was ruled out using platelets as reference cells: no platelet aggregation pattern and no increase in intracellular calcium were observed.