The principally active hemolytic toxin (cardiotoxin) previously purified from the venom of the Thailand cobra, Naja naja siamensis, was shown to produce spontaneous twitching, contractures and membrane depolarization in sartorius muscles from the frog, Rana pipiens. Spontaneous twitching, observed at concentrations greater than 0.1 uM was completely abolished by addition of tetrodotoxin and not affected by d-tubocurarine. Dose and time dependent membrane depolarization of muscle fibers was observed to occur within 10-30 min at 0.2 to 1.0 uM concentrations of the toxin. These observations, taken together with an amino acid analysis characteristic of previously described cobra venom cardiotoxins, characterized this hemolytic toxin as a cardiotoxin. In the absence of EDTA the initial velocities of erythrocyte hemolysis for this toxin showed a sigmoidal concentration dependence which became hyperbolic in the presence of EDTA. The largest increases in hemolysis rates on addition of 1 mM EDTA were observed at low toxin concentrations. In the presence of EDTA extracellular and membrane associated divalent cations are complexed, thus alleviating their competition with toxin for binding to the membrane, a key and apparently rate-determining initial step which leads to hemolysis. In the presence of EDTA hemolysis rates increased linearly at low toxin concentration and reached an extrapolated maximum value at toxin concentrations at which, given its molecular dimensions, there are just sufficient toxin molecules to cover the entire membrane surface area provided by the erythrocytes.