High-level resistance to aminoglycosides in enterococci often is mediated by aminoglycoside-modifying enzymes, and the corresponding genes generally are located on self-transferable plasmids. These enzymes are similar to those in staphylococci but differ from the modifying enzymes of gram-negative bacteria. Three classes of enzymes are distinguished, depending upon the reaction catalyzed. All but amikacin and netilmicin confer high-level resistance to the antibiotics that are modified in vitro. However, the synergistic activity of these last two antibiotics in combination with beta-lactam agents can be suppressed, as has always been found in relation to high-level resistance to the aminoglycosides. Acquisition of glycopeptide resistance by enterococci recently was reported. Strains of two phenotypes have been distinguished: those that are resistant to high levels of vancomycin and teicoplanin and those that are inducibly resistant to low levels of vancomycin and susceptible to teicoplanin. In strains of Enterococcus faecium highly resistant to glycopeptides, we have characterized plasmids ranging from 34 to 40 kilobases that are often self-transferable to other gram-positive organisms. The resistance gene vanA has been cloned, and its nucleotide sequence has been determined. Hybridization experiments showed that this resistance determinant is present in all of our enterococcal strains that are highly resistant to glycopeptides. The vanA gene is part of a cluster of plasmid genes responsible for synthesis of peptidoglycan precursors containing a depsipeptide instead of the usual D-alanyl-D-alanine terminus. Reduced affinity of glycopeptides to these precursors confers resistance to the antibiotics.