The structure of a new antibiotic of the iturin group, bacillomycin F, has been demonstrated. It is a mixture of homologous peptidolipids, essentially C51H80N12O14 and C52N82N12O14. The lipid moiety consists of minor isoC15, anteisoC15 beta-amino acids and major isoC16, isoC17 and anteisoC17 beta-amino acids. The peptide sequence was determined by studying the peptides obtained after mild HCl hydrolysis and by Edman degradation of bacillomycin F treated with N-bromosuccinimide. The sequence was confirmed by two-dimensional NMR spectrometry and fast-atom-bombardment mass spectrometry gave the molecular masses of the homologous compounds. Bacillomycin F is a cyclic peptidolipid; its complete structure is given in the paper.