Three of five natural plasmids carrying a wild-type vanA gene cluster did not confer LY333328 glycopeptide resistance on Enterococcus faecalis JH2-2 (MIC = 2 μg/ml). The two remaining plasmids conferred resistance to the drug (MIC, 8 μg/ml). The vanB gene cluster did not confer resistance to LY333328, since this antibiotic was not an inducer. Mutations in the vanSB sensor gene that allowed induction by teicoplanin or constitutive expression of the vanB cluster led to LY333328 resistance (MIC, 8 to 16 μg/ml). Overproduction of the VanH, VanA, and VanX proteins for d-alanyl-d-lactate (d-Ala-d-Lac) synthesis and d-Ala-d-Ala hydrolysis was sufficient for resistance to LY333328 (MIC, 16 μg/ml). Mutations in the host d-Ala:d-Ala ligase contributed to LY333328 resistance in certain VanA- and VanB-type strains, but the MICs of the antibiotic did not exceed 16 μg/ml. Addition of d-2-hydroxybutyrate in the culture medium of mutants that did not produce the VanH d-lactate dehydrogenase led to incorporation of this d-2-hydroxy acid at the C-terminal ends of the peptidoglycan precursors and to LY333328 resistance (MIC, 64 μg/ml). The vanZ gene of the vanA cluster conferred resistance to LY333328 (MIC, 8 μg/ml) by an unknown mechanism. These data indicate that VanA- and VanB-type enterococci may acquire moderate-level resistance to LY333328 (MIC ≤ 16 μg/ml) in a single step by various mechanisms.