Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Na(v)s) at two pharmacologically distinct sites. Alpha toxins bind at receptor site-3 and inhibit channel inactivation, whereas beta toxins bind at receptor site-4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and ability to compete for the receptor sites at Na(v) subtypes. To elucidate the toxin-channel surface of interaction at both receptor sites and clarify the molecular basis of varying toxin preferences, an efficient bacterial system for their expression in recombinant form was established. Mutagenesis accompanied by toxicity, binding and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography uncovered a bipartite bioactive surface in toxin representatives of all pharmacological groups. Exchange of external loops between the mammalian brain channel rNa(v)1.2a and the insect channel DmNa(v)1 highlighted channel regions involved in the varying sensitivity to assorted toxins. In parallel, thorough mutagenesis of channel external loops illuminated points of putative interaction with the toxins. Amino acid substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain II of rNa(v)1.2a had prominent impact on the activity of the beta-toxin Css4 (from Centruroides suffusus suffusus), and substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain IV affected the activity of the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus). Rosetta modeling of toxin-Na(v) interaction using the voltage sensor module of the potassium channel as template raises commonalities in the way alpha and beta toxins interact with the channel. Css4 interacts with rNa(v)1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain II, while Lqh2 interacts with rNa(v)1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain IV. Double-mutant cycle analysis and dissociation assays employing a battery of Lqh2 mutants against rNa(v)1.2a mutants identified the docking orientation of alpha toxins at the channel external surface of the Gating-module in domain IV. The other point of interaction between the toxin and the channel has not yet been defined and may involve channel residues of either the Pore-module or the Gating-module.