The conformation of viroisin, the monocyclic toxic heptapeptide of the virotoxin family from mushroom Amanita virosa, was analysed using two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics simulations. All proton signals were completely assigned, and interproton distances were determined using data from rotating-frame nuclear Overhauser enhancement. The backbone dihedral angles were deduced from measurements of coupling constants. The temperature dependence of the amide proton chemical shifts provided information about hydrogen bonding. Six probable solution conformations of viroisin were derived from the use of distance geometry and restrained molecular dynamics based on a set of distance constraints obtained from experimental data. The results of the structural analysis indicate that viroisin has a well ordered conformation in solution. In all these conformations the functional groups essential for toxicity orient themselves in the same direction so as to bind to the target proteins. This feature is consistent with previous results about the formation of a hydrophobic pocket on one side of the molecule.