The vanR, vanS, vanH, vanA, and vanX genes of enterococcal transposon Tn1546 were introduced into the chromosome of Enterococcus faecalis JH2-2. Complementation of this portion of the van gene cluster by a plasmid encoding VanY D,D-carboxypeptidase led to a fourfold increase in the vancomycin MIC (from 16 to 64 micrograms/ml). Multicopy plasmids pAT80 (vanR vanS vanH vanA vanX) and pAT382 (vanR vanS vanH vanA vanX vanY) conferred similar levels of vancomycin resistance to JH2-2. The addition of D-alanine (100 mM) to the culture medium restored the vancomycin susceptibility of E. faecalis JH2-2/pAT80. The pentapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala partially replaced pentadepsipeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac when the strain was grown in the presence of D-alanine. In contrast, resistance mediated by pAT382 was almost unaffected by the addition of the amino acid. Expression of the vanY gene of pAT382 resulted in the formation of the tetrapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala, indicating that a portion of the cytoplasmic precursors had been hydrolyzed. These results show that VanY contributes to glycopeptide resistance in conditions in which pentapeptide is present in the cytoplasm above a threshold concentration. However, the contribution of the enzyme to high-level resistance mediated by Tn1546 appears to be moderate, probably because hydrolysis of D-alanyl-D-alanine by VanX efficiently prevents synthesis of the pentapeptide.