A 600-MHz proton NMR study of natural charybdotoxin, a toxin acting on K+ channels, is reported. The unambiguous sequential assignment of all the protons of the toxin was achieved. The analysis of NOEs and of backbone coupling constants showed the existence of an α-helix (residues 10–19) and of an antiparallel β-sheet in the 26–35 part. Three-dimensional structures were generated by distance geometry, using a set of 114 inter-residual calibrated constraints (63 sequential, 47 medium and long range, 4 hydrogen bonds) and 29 π angles. These structures show that charbdotoxin is composed of a β-sheet linked to an α-helix by two disulphide bridges and to an extended fragment by the third disulphide bridge. Comparison with the other known structures of long and short scorpion toxins shows that this structural motif is common to all these proteins.