The action of 7.2 microM cardiotoxin on 0.25% human erythrocytes in a plasma extender solution was studied by the interaction of toxin with intact red blood cells and subsequent hemolysis of the cells. The binding of toxin to cells was completed within 10 min, whereas the membrane rigidity was weakened in a non-lytic period for about 25 min. The toxin molecules bound almost exclusively to the membrane. The bound toxin could not be liberated with either 0.5% Triton X-100 or 0.1 N NaOH. The degree of binding was slightly reduced in the presence of 10 mM mono- and divalent inorganic salts. The action of toxin might weaken the in situ association of several proteins that are linked with band 3 protein of the membrane, thus making the cells fragile and altering the shape of the cell to a smooth sphere.