The high-resolution three-dimensional solution structure of the plant toxin hordothionin-alpha obtained from korean barley was determined by using two-dimensional NMR techniques combined with distance geometry and restrained molecular dynamics. Experimentally derived restraints including 292 interproton distances from nuclear Overhauser effect measurements, 16 hydrogen bond restraints together with four disulphide bridge restraints were used as input to calculations of distance geometry and restrained molecular dynamics. Also included in the calculations were 36 phi and 17 chi 1 torsion angles obtained from 33JHN alpha and 3J alpha beta coupling constants in double quantum filtered COSY and primitive exclusive COSY experiments, respectively. The overall protein fold is similar to crambin and purothionin-alpha 1. Two alpha-helices running in opposite directions are found on the basis of 3JHN alpha and 3J alpha beta and deuterium exchange rates for backbone NH protons, and encompass residues 7-18 and 22-28. These two helices are connected by a turn and form a 'helix-turn-helix' motif. A short stretch of an anti-parallel beta-sheet exists between residues 1-4 and 31-34. the two protein termini of hordothionin-alpha are 'well-anchored'; the N-terminus of the protein is immobilized by this short beta-sheet whereas the C-terminus is 'pasted' to the carbonyl group of Cys-4 by a very stable hydrogen bond. The average root-mean-square differences for the backbone and heavy atoms after the restrained molecular dynamics calculations are 0.62 and 1.16 A respectively. These numbers represent a significant improvement over the corresponding values for the previous NMR structures of other thionins. The distance violation from the experimental interproton distances for the final structures is 0.14 for all atoms.