In the course of a screening aimed at new metabolites, we used electrospray mass spectrometry (ESI-MS, CID-MS/MS)1,2) for the detection of linear peptides and peptaibols in microbial culture extracts3~5). Here we report the isolation and structure elucidation of peptaibolin (1, Fig. 1) as an unusual representative of the peptaibol family of lipophilic antibiotics.1 was disclosed in two fungal strains, Sepedonium sp. HKI-0117 and Sepedonium ampullosporum HKI-0053 coproducing the neuroleptic peptaibol-type antibiotic ampullosporin5). In contrast to the known peptaibols6), the compound 1 contains only five peptide bonds. However, it displays all of the structural features of the peptaibols such as the presence of α-aminobutyric acid moieties, of an acylated amino terminus, and an alcoholic group at the opposite end of the peptide chain.