In a recent note to Nature, R. MacKinnon has raised the possibility that potassium channel gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes and that by increasing their partial concentration adjacent to their receptor, they affect channel function with apparent high affinity (Lee and MacKinnon (2004) Nature 430, 232-235). This suggestion was adopted by Smith et al. (Smith, J. J., Alphy, S., Seibert, A. L., and Blumenthal, K. M. (2005) J. Biol. Chem. 280, 11127-11133), who analyzed the partitioning of sodium channel modifiers in liposomes. They found that certain modifiers were able to partition in these artificial membranes, and on this basis, they have extrapolated that scorpion β-toxins interact with their channel receptor in a similar mechanism as that proposed by MacKinnon. Since this hypothesis has actually raised a new conception, we examined it in binding assays using a number of pharmacologically distinct scorpion β-toxins and insect and mammalian neuronal membrane preparations, as well as by analyzing the rate by which the toxin effect on gating of Drosophila DmNav1 and rat brain rNav1.2a develops. We show that in general, scorpion β-toxins do not partition in neuronal membranes and that in the case in which a depressant β-toxin partitions in insect neuronal membranes, this partitioning is unrelated to its interaction with the receptor site and the effect on the gating properties of the sodium channel. These results negate the hypothesis that the high affinity of β-toxins for sodium channels is gained by their ability to partition in the phospholipid bilayer and clearly indicate that the receptor site for scorpion β-toxins is accessible to the extracellular solvent.