Over a 10-month period, 22 beta-lactamase negative E. faecium strains resistant to ampicillin were isolated from severely compromised hospitalized patients. Most isolates were clinically significant. Twenty of these strains were also resistant to extraordinarily high levels of gentamicin, a finding described rarely in E. faecium. By whole-cell DNA restriction endonuclease digestion, the 20 strains with both ampicillin and high-level gentamicin resistance segregated into only 3 different groups, suggesting introduction of limited clones into this population of patients. Plasmid DNA profiles and plasmid DNA restriction enzyme analysis supported this grouping for 18 of these 20 strains, the two remaining isolates had slightly different profiles. Both strains lacking high-level gentamicin resistance had clearly different molecular profiles. The results of hybridization experiments strongly suggested the presence of a similar genetic determinant of high-level gentamicin resistance in E. faecalis and E. faecium. High-level gentamicin resistance in E. faecium was shown to be transferable on conjugative plasmids, so that further dissemination of this resistance trait may be anticipated. Our data indicate that the use of a nucleic acid probe is a promising diagnostic tool for screening both E. faecium and E. faecalis for high-level gentamicin resistance.