The novel peptide lactone hormaomycin produced by Streptomyces griseofavus is selectively active against some gram-positive bacteria and influences aerial mycelium formation as well as secondary metabolite production. This study reports the elucidation of its structure. Hormaomycin (C₄₁H₅₉ClN₁₀O₁₁) contains known amino acids allo-threonine (a-Thr), isoleucine (Ile) and 3-methylphenylalanine (Phe(3-Me)), as well as new amino acids 4-[(Z)-propenyl]proline (Pro(4-Pe)) and 3-(2-nitrocyclopropyl)alanine (Ala(3-Ncp)) (with Phe(3-Me) and Ala(3-Ncp) each occurring twice). The stereochemistry of known amino acids was determined via acidic total hydrolysis, esterification/acetylation derivatization, and chromatographic analysis as D-allo-threonine, L-isoleucine, and D-threo-(3-methyl)phenylalanine (two molecules). The amino acid participating in the C-terminal lactone bonding was identified by LiAlH₄ reduction and GC/MS analysis of the hydrolyzed product. Sequence analysis combined results from partial hydrolyzates (tripeptides identified by GC/MS coupling), FAB mass spectrometry of the open-chain hormaomycin methyl ester (confirming sequence via successive fragment cleavage), and ROESY-NMR experiments (verifying partial sequence). The combined data yielded constitution 3 for hormaomycin, a peptide lactone structural type not hitherto described as a microbial secondary metabolite.