Sea anemone actinoporins constitute a protein family of multigene pore–forming toxins (PFT). Equinatoxin II (EqtII), fragaceatoxin C (FraC), and sticholysins I and II (StnI and StnII), produced by three different sea anemone species, are the only actinoporins whose molecular structures have been studied in depth. These four proteins show high sequence identities and practically coincident three–dimensional structures. However, their pore–forming activity can be quite different depending on the model lipid system employed, a feature which has not been systematically studied before. Therefore, the aim of this work was to evaluate and compare the influence of several distinct membrane conditions on their particular pore–forming behavior. Using a complex model membrane system, such as sheep erythrocytes, StnII showed much higher hemolytic activity than the other three actinoporins studied. In lipid-model systems, pore–forming ability decreased in the order of StnI > StnII > EqtII > FraC when assayed against DOPC:SM (4:1) vesicles, with the membrane binding being the rate–limiting step. When using DOPC:SM:Chol (1:1:1) LUVs, the presence of Chol not only enhanced membrane binding affinities by about two orders of magnitude but also revealed how StnII was much faster than the other three actinoporins in producing calcein release. This ability agrees with the proposal that explains this behavior in terms of their high sequence variability along their first 30 N–terminal residues. The influence of interfacial hydrogen bonding in SM or dihydro–SM containing bilayers was also shown to be a generalized feature of the four actinoporins studied. It is finally hypothesized that this observed variable ability could be explained as a consequence of their distinct specificities and/or membrane binding affinities. Eventually, this behavior can be modulated by the nature of their natural target membranes or the interaction with not-yet characterized isotoxin forms from the same sea anemone species.