AbstractA cell free system fromStaphylococcus aureus52A5 which incorporates UDPMurNAc‐L‐ala‐D‐isoglu‐L‐lys‐D‐ala‐14C‐D‐ala into lipid intermediates and peptidoglycan is described. During peptidoglycan synthesis, glycine binding to the lipid intermediates and peptidoglycan is also demonstrated as well as release of the terminal D‐alanine from the pentapeptide moiety. This release of D‐alanine is strongly