The prevalence of resistance to high levels of gentamicin among 182 isolates of Enterococcus faecalis from 2 Iranian hospitals was 42%. Gentamicin resistance was associated with conjugative plasmids (>70 kb) in most strains. Fingerprinting using EcoRI and HindIII showed genetic variation among these plasmids and gave evidence of nosocomial outbreaks and persistence of infection in different wards of the study hospitals, as well as transfer of plasmids between genetically diverse isolates. Using EcoRI, hospital-based specific plasmid fingerprints were detected for the isolates that had previously proved to be unrelated by multilocus enzyme electrophoresis, suggesting the persistence of related plasmids at each hospital, though minor changes in these related plasmids could be detected with HindIII.