Dehydroamino acids are valuable building blocks that are a challenge to incorporate synthetically into unprotected peptides. Lantibiotic synthetases possess dehydration activity that converts Ser and Thr residues in their peptide substrates into dehydroalanine and dehydrobutyrine residues, respectively. We show here that lacticin 481 synthetase can convert the Thr analogs (R)-3-EtSer, (R)-3-vinylSer, (R)-3-ethynylSer, and (R)-3-[(E)-propenyl]Ser into the corresponding dehydro amino acids when incorporated into its peptide substrate. This relaxed substrate specificity holds promise for using the enzyme for synthetic purposes and for lantibiotic engineering. On the other hand, (R)-3-PrSer, (R)-3-iPrSer, and allo-Thr are not substrates for the enzyme.