Hypelcin A is a mixture of antibiotic peptides produced by Hypocrea peltata. Hypelcin A-I, A-II, A-III, A-IV, A-V, A-VI, A-VII, A-VIII and A-IX are components of this mixture purified by reversed-phase high-performance liquid chromatography. The amino acid sequences of these peptides were determined by fast-atom bombardment mass spectrometry and fast-atom bombardment mass spectrometry/mass spectrometry with the help of NMR spectroscopy. The relative molecular masses of these peptides were all ~2000 and their structures were very similar. Hypelcin A is isolated from Hypocrea peltata (Jungh) Sacc. which prevents the growth of Lentinus edodes, a Japanese edible mushroom. Hypelcin A belongs to the class of peptaibols having the following structural features: a richness in helix-promoting 7-aminoisobutyric acid (Aib); the presence of two proline residues; the C-terminal is linked with an amino alcohol, leucinol (Lol); and the compounds are acetylated at the N-terminal. Peptaibols, which can also be regarded as alamethicin analogues, show membrane-modifying properties which give rise to various bioactivities, such as formation of voltage-gated ion channels, haemolysis, and membrane fusion. Suzukacillins, paraselcins, trichosporins, antiamoebins, trichorzianines, trichotoxins, emerimicins and yervamicins are some peptaibols with similar structures and activities. Hypelcin A, which inhibits growth of various fungi and bacteria, has been shown to uncouple oxidative phosphorylation in rat liver mitochondria and to induce permeability changes in phosphatidylcholine bilayers as a result of it being a mixture of components. In an attempt to separate and identify the components of hypelcin A, we previously reported22 the isolation and structure investigation of hypelcin A-I, A-II, A-III and A-IV, where hypelcin A-III and A-IV could only be purified partially and the complete structure of hypelcin A-III could not be clarified. In this study, further purification and separation of the above mentioned hypelcins were performed by repeated high-performance liquid chromatography (HPLC) and five new compounds, hypelcin A-V, A-VI, A-VII, A-VIII and A-IX, were isolated. The previously reported structures of hypelcin A-I, A-II, A-III and A-IV were reinvestigated and the structures of the new ones were elucidated by fast-atom bombardment mass spectrometry (FAB-MS), fast-atom bombardment mass spectrometry/mass spectrometry (FAB-MS/MS) and 2D NMR techniques.