Abstract Two-component lantibiotics comprise of two post-translationally modified peptides that synergistically exert antimicrobial activity. Most known two-component lantibiotics are made up of a structurally conserved α-lanthipeptide that binds to the peptidoglycan precursor lipid II and a β-lanthipeptide that interacts with the α-peptide-lipid II complex to disrupt the membrane of susceptible bacteria. A few two-component lantibiotics including roseocin produced by Streptomycetes do not follow this general scheme and act by different, currently unresolved mechanisms. An important first step in studying this latter group of lanthipeptides is determination of their chemical structure. Roseocin’s β-peptide (Rosβ) is formed by the RosM lanthipeptide synthetase from the RosA1 precursor peptide. RosM carries out nine dehydrations of Ser and Thr residues to the corresponding dehydroamino acids followed by six Michael-type additions of the thiols of Cys residues to a subset of the dehydroamino acids to form six thioether crosslinks. Sequence alignment with other structurally characterized lanthipeptides does not allow prediction of the ring pattern of Rosβ. In this study, Rosβ was produced in Escherichia coli and its ring pattern was established by multi-dimensional NMR spectroscopy. The stereochemistry of the lanthionine and methyllanthionine residues was determined by Marfey’s analysis with authentic standards. Rosβ is shown to have a unique ring pattern amongst previously characterized lanthipeptides. TOC graphic