Lactocin S is a lantibiotic peptide with potent antibacterial activity against a range of Gram-positive bacteria. Because of challenges in obtaining sufficient quantities of this compound from natural Sources, the stereochemistry of the lanthionine residues in lactocin S had not been confirmed. This report describes the chemical synthesis of lactocin S on chlorotrityl polystyrene resin in 10% overall yield using intramolecular cyclization to form the lanthionine rings and employing fragment coupling for the two N-terminal residues. This represents the first report of solid-supported synthesis of a naturally occurring lantibiotic. Comparison to lactocin S isolated from Lactobacillus sakei L45 using a combination of HPLC, MS/MS sequencing, bacteria] testing, and chiral GC-MS analysis confirmed the initially proposed structure and the stereochemistry of the dl-lanthionine residues.