A total of 1,799 Enterococcus faecalis isolates were isolated from inpatients of Gunma University Hospital, Gunma, Japan, between 1992 and 1996. Four hundred thirty-two (22.3%) of the 1,799 isolates had high-level gentamicin resistance. Eighty-one of the 432 isolates were classified and were placed into four groups (group A through group D) with respect to the EcoRI restriction endonuclease profiles of the plasmid DNAs isolated from these strains. The 81 isolates were isolated from 36 patients. For 35 of the 36 patients, the same gentamicin-resistant isolates were isolated from the same or different specimens isolated from the same patient at different times during the hospitalization. For one other patient, two different groups of the isolates were isolated from the same specimen. Groups A, B, C, and D were isolated from 5, 14, 12, and 6 patients, respectively. The strains had multiple-drug resistance. The restriction endonuclease digestion patterns of the E. faecalis chromosomal DNAs isolated from isolates in the same group were also identical. The patients who had been infected with the gentamicin-resistant isolates from each group were geographically clustered on a ward(s). These results suggest that the isolates in each group were derived from a common source and had spread in the ward. The gentamicin-resistant isolates exhibited a clumping response upon exposure to pheromone (E. faecalis FA2-2 culture filtrate). The gentamicin resistance transferred at a high frequency to the recipient E. faecalis isolates by broth mating, and the pheromone-responsive plasmids encoding the gentamicin resistance were identified in these isolates.