The solution conformations of protodestruxin, desmethyldestruxin B and destruxin B, insecticidal cyclodepsipeptides, in DMSO-d6 have been studied through the use of 100-MHz proton NMR spectroscopy. Proton-deuteron exchange and temperature dependence of the peptide proton chemical shift were used to delineate peptide protons in terms of exposure to solvent. The results defined the β-alanine and isoleucine peptide protons of desmethylde-struxin B and destruxin B as solvent-shielded. A rigid conformation of desmethyldestruxin B and destruxin B, in which there are two hydrogen-bonded rings of ten atoms, is proposed. Protodestruxin seems to have a mobile conformation in DMSO-d6. The results indicate that the biological N-methylation of the alanine peptide proton in protodestruxin decreases the number of possible conformations which are stabilized by intramolecular hydrogen bonding and, as the result, fixes the molecule in a specific conformation.