<jats:p> The ≈80-kb <jats:italic>pksX</jats:italic> gene cluster in <jats:italic>Bacillus subtilis</jats:italic> encodes an unusual hybrid polyketide/nonribosomal peptide synthase that has been linked to the production of the uncharacterized antibiotic bacillaene. Multiple copies of this synthase, each similar in size to the ribosome, assemble into a single organelle-like complex with a mass of tens to hundreds of megadaltons. The resource requirements of the assembled megacomplex suggest that bacillaene has an important biological role. By coupling a differential NMR spectroscopic technique with genetically manipulated strains of <jats:italic>B. subtilis</jats:italic> , we were able to characterize the structure of this unusual secondary metabolite, which could not be predicted by using bioinformatic analysis. We report that bacillaene is a linear molecule with two amide bonds: the first links an α-hydroxy carboxylic acid to a ω-amino carboxylic acid containing a conjugated hexaene, and the second links the hexaene-containing carboxylic acid to an (ω-1) amino carboxylic acid containing a conjugated triene. Knowledge of bacillaene's structure has enabled us to annotate the <jats:italic>pksX</jats:italic> gene cluster and should facilitate the study of bacillaene's biosynthesis as well as its biological role in <jats:italic>B. subtilis</jats:italic> .