<jats:title>Abstract</jats:title><jats:p>Feglymycin, a peptide antibiotic produced by <jats:italic>Streptomyces sp</jats:italic>. DSM 11171, consists mostly of nonproteinogenic phenylglycine‐type amino acids. It possesses antibacterial activity against methicillin‐resistant <jats:italic>Staphylococcus aureus</jats:italic> strains and antiviral activity against HIV. Inhibition of the early steps of bacterial peptidoglycan synthesis indicated a mode of action different from those of other peptide antibiotics. Here we describe the identification and assignment of the feglymycin (<jats:italic>feg</jats:italic>) biosynthesis gene cluster, which codes for a 13‐module nonribosomal peptide synthetase (NRPS) system. Inactivation of an NRPS gene and supplementation of a hydroxymandelate oxidase mutant with the amino acid <jats:sc>l</jats:sc>‐Hpg proved the identity of the <jats:italic>feg</jats:italic> cluster. Feeding of Hpg‐related unnatural amino acids was not successful. This characterization of the <jats:italic>feg</jats:italic> cluster is an important step to understanding the biosynthesis of this potent antibacterial peptide.