Antimicrobial susceptibilities and DNA contents were analyzed for six clinical isolates of Enterococcus faecalis that had high-level resistance to gentamicin (MIC > 2,000 micrograms/ml) but not streptomycin and were obtained from patients in diverse geographic areas. Contour-clamped homogeneous electric field electrophoresis of genomic DNA showed all isolates to be different strains. Gentamicin resistance was transferred from four isolates to plasmid-free enterococcal recipients in filter matings. Restriction enzyme analysis of transconjugants showed distinct gentamicin resistance plasmids. A probe specific for the gentamicin resistance determinant hybridized to the plasmids of four isolates and to the chromosomes of two isolates. These findings suggest that clonal dissemination is not responsible for the spread of these resistant strains, that resistance determinants occur on different plasmids as well as on the chromosome of E. faecalis, and that the genetic determinants of resistance are related.