Between November 1981 and October 1984, 48 of 3,456 clinical isolates of Streptococcus faecalis that were studied showed high-level (greater than 2,000 micrograms/ml) resistance to gentamicin. A case-control study comparing patients with gentamicin-susceptible (MIC less than 64 micrograms/ml) and -resistant S. faecalis infections showed significant associations (P less than .01) between the development of infection with highly gentamicin-resistant S. faecalis and prior antimicrobial therapy (particularly with cephalosporins or aminoglycosides), perioperative antibiotic prophylaxis, prior surgical procedures, and longer hospitalization. All highly resistant strains appeared to be nosocomial since 12 cases were clustered on a surgical floor and in a burn unit. In vitro transfer of gentamicin resistance by filter mating was observed for 44 of 48 isolates. The use of plasmid content as an epidemiological marker suggested nosocomial transmission and exogenous acquisition of S. faecalis.