Enterococci are important nosocomial pathogens among which resistance to multiple antibiotics is being recognized with increasing frequency. We characterized three clinical isolates from three New York City hospitals that demonstrated concomitant resistance to vancomycin (one VanA, two VanB phenotypes) and high-level resistance to penicillin. Two Enterococcus faecium strains were intrinsically highly resistant to penicillin and showed very low affinity for penicillin of penicillin-binding protein 5. Unlike previously described glycopeptide-resistant enterococci, these strains were not hypersusceptible to beta-lactam agents after vancomycin induction, and combinations of penicillin and vancomycin were not synergistic against them. A third isolate, Enterococcus faecalis, produced beta-lactamase. Two of the three strains were also highly resistant to all aminoglycosides. Emergence of concomitant high-level resistance to multiple antibiotic classes among enterococci considerably narrows the therapeutic options for treatment of infections due to these opportunistic pathogens.