The lantibiotics are a class of ribosomally synthesized and posttranslationally modified peptide antibiotics containing the thioether cross-links lanthionine (Lan) and 3-methyllanthionine (MeLan) and typically also the dehydroamino acids dehydroalanine (Dha) and (Z)-dehydrobutyrine (Dhb). The modifications are formed by dehydration of Ser/Thr residues to produce the Dha and Dhb structures, and subsequent conjugate additions of Cys residues onto the unsaturated amino acids to form thioether rings (Lan and MeLan). Because of their ribosomal origin, investigations of the substrate specificity of lantibiotic synthetases have typically focused on site-directed mutagenesis. With the in vitro reconstitution of lacticin 481 synthetase, its substrate specificity was explored in much greater detail by the incorporation of a series of nonproteinogenic Ser, Thr, and Cys analogues into a truncated prelacticin peptide LctA using a combination of solid-phase peptide synthesis (SPPS) and expressed protein ligation (EPL). The strategy described can be used for the growing number of ribosomally synthesized and posttranslationally modified natural products such as the microcins and patellamides.