Amino acid analysis on the acid hydrolyzate of polymyxin S1 revealed its amino acid composition. Isolation of the constituent amino acids and measurement of their optical activities clarified their chiralities: Dab(5L), Thr(3L), Ser(1D) and Phe(1D). The constituent fatty acid was identified with anteisononanoic acid by gas chromatography and mass spectrometry. By the action of polymyxin acylase, deacyl polymyxin S was obtained. Successive EDMAN degradation reaction on deacyl polymyxin S revealed the amino acid sequence. Further evidence for the structure of polymyxin S1 was obtained by partial acid hydrolysis on tetra-(DNP)-polymyxin S1.