Previous studies show that membrane-damaging activity of cardiotoxins (CTXs) is associated with their binding with anionic phospholipids on membrane. In order to examine this proposition, studies on the interaction between Taiwan cobra CTXs and zwitterionic phospholipids were conducted in the present study. Although membrane-damaging activity of CTX2 and CTX4 was affected by phospholipid compositions, CTX2 and CTX4 were able to induce membrane permeability of zwitterionic phospholipid vesicles. CTX2 showed a higher membrane-damaging activity toward egg yolk phosphatidylcholine (EYPC), EYPC/egg yolk phosphatidylethanolamine (EYPE) or EYPC/egg yolk phosphatidylglycerol (EYPG) than CTX4 did. Moreover, CTX2- and CTX4-induced membrane permeability of EYPC and EYPC/EYPE vesicles was higher than that of EYPC/EYPG vesicles. CTX2 and CTX4 displayed different membrane-bound mode in binding with EYPC, EYPC/EYPE and EYPC/EYPG vesicles as revealed by N-( fluorescein - 5 - thiocarbamoyl) - 1, 2 - dihexadecanoyl-phosphatidyl ethanolamine fluorescence enhancement, color transformation of phospholipids/polydiacetylene membrane assay and rhodamine-self quenching assay. CTX2 and CTX4 showed a similar binding affinity with phospholipid vesicles, but the reversibility in binding with phospholipid vesicles differed between CTX2 and CTX4. Compared with that of CTX4, membrane-damaging activity of CTX2 was markedly inhibited by anti-CTX antibodies. Taken together with the finding that gross conformation of CTX2 and CTX4 differs, our data suggest that membrane-damaging activity of CTX2 and CTX4 critically depends on their membrane-bound mode and is affected by phospholipid composition. Moreover, the ability of CTX to induce membrane permeability is not exclusively attributed to the binding with anionic phospholipids.