The structure of mycosubtilin, a peptidolipid antibiotic from Bacillus subtilis, was revised by FAB mass spectrometry, 2D NMR spectrometry and also by Edman degradation of the peptide resulting from the N-bromosuccinimide reaction. Four homologous IS-amino acid components were identified by capillary gas chromatography. The cyclopeptide mycosubtilin consists of seven a-amino acids in an LDDLLDL sequence closed by a s-amino acid linkage similar to that found in other antibiotics of the iturin group.