A comparison was made of the binding modes of the bacterial cell wall precursor L-lysyl-D-alanyl-D-alanine to the glycopeptide antibiotic vancomycin and to the D-alanyl-D-alanine-cleaving peptidase of Streptomyces sp. strain R61, a model for cell wall-synthesizing enzymes whose X-ray three-dimensional structure is established. In each of the two pairings (vancomycin with peptide and DD-peptidase with peptide), polypeptide backbones were antiparallel, and the antibiotic or enzyme enveloped the peptide substrate from opposite sides. Hydrogen-bonding groups on the substrate which are involved with the DD-peptidase were shown to be different from the ones reported from nuclear magnetic resonance studies to be involved with vancomycin. Because of steric hindrance, the binding of either molecule to the substrate prevents the binding of the other molecule. Binding to the substrate by a D-alanyl-D-alanine-recognizing protein in a manner similar to that used by the DD-peptidase could explain recent observations of vancomycin resistance, in which a new membrane-associated protein has been detected.