The solution structure of a recombinant active α-neurotoxin from Leiurus quinquestriatus hebraeus, LqhαIT, was determined by proton two- dimensional nuclear magnetic resonance spectroscopy (2D NMR). This toxin is the most insecticidal among scorpion α-neurotoxins and, therefore, serves as a model for clarifying the structural basis for their biological activity and selective toxicity. A set of 29 structures was generated without constraint violations exceeding 0.4 Å. These structures had root mean square deviations of 0.49 and 1.00 Å with respect to the average structure for backbone atoms and all heavy atoms, respectively. Similarly to other scorpion toxins, the structure of LqhαIT consists of an α-helix, a three-strand antiparallel β- sheet, three type I tight turns, a five-residue turn, and a hydrophobic patch that includes tyrosine and tryptophan rings in a 'herringbone' arrangement. Positive φ angles were found for Ala50 and Asn11, suggesting their proximity to functionally important regions of the molecule. The sample exhibited conformational heterogeneity over a wide range of experimental conditions, and two conformations were observed for the majority of protein residues. The ratio between these conformations was temperature-dependent, and the rate of their interconversions was estimated to be on the order of 1- 5 s-1 at 308 K. The conformation of the polypeptide backbone of LqhαIT is very similar to that of the most active antimammalian scorpion α-toxin, AaHII, from Androctonus australis Hector (60% amino acid sequence homology). Yet, several important differences were observed at the 5-residue turn comprising residues Lys8-Cys12, the C-terminal segment, and the mutual disposition of these two regions. 2D NMR studies of the R64H mutant, which is 3 times more toxic than the unmodified LqhαIT, demonstrated the importance of the spatial orientation of the last residue side chain for toxicity of LqhαIT.