Amino acid analysis of the acid hydrolyzate of octapeptin D revealed the amino acid composition. These amino acids were converted to L-leucyl-derivatives and analyzed by high performance liquid chromatography to clarify their chiralities. These were determined to be: 2,4-diaminobutyric acid (4L), Ser (D), Leu (2L, 1D). Deacylation with polymyxin acylase afforded deacyl octapeptin D. EDMAN degradation on deacyl octapeptin D revealed the N-terminal amino acid. Application of the chemical cleavage reaction for fragmentation of seryl peptides on tri (DNP)-octapeptin D afforded a DNP-heptapeptide, whose sequence was clarified by EDMAN degradation. Octapeptin D was separated into four components (D1,D2,D3 and D4) by high performance liquid chromatography. All the components were examined for their amino acid and fatty acid compositions. From the results, the structures of octapeptins D1, D2, D3 and D4 were determined.