Mycosubtilin, an antifungal agent isolated from Bacillus subtilis is a mixture of homologous lipopeptides essentially C54H83N14O16 and C55H85N15O16. The differences in their structures was found in the lipid moiety which contains several beta-amino acids; the structure of these beta-amino acids was demonstrated by combined gas chromatography-mass spectrometry of the N-trifluoroacetyl n-butyl esters; a strong peak at m/e = 240 indicates a beta-amino group. The comparison of the derivatives of natural amino acids with synthetic 3-aminohexadecanoic and 3-aminoheptadecanoic acids indicates that natural beta-amino acids are a mixture of 3-amino-14-methylpentadecanoic acid (35%), 3-amino-14-methylhexadecanoic acid (59%), 3-aminohexadecanoic acid (6%) and a trace of C18 beta-amino acid. The peptide moiety contains 8 moles of amino acids, two of D-aspargine, two of L-asparagine, and one of L-glutamine, L-proline, D-serine and D-tyrosine. The peptide sequence was determinated by partial acid hydrolysis of mycosubtilin and isolation and structural determination of the peptides from the hydrolysates. Four liposoluble peptides and four hydrosoluble peptides were studied. The results gave the cyclic structure shown on Formula 1 for mycosubtilin.