To identify any change in the antibiotic resistance of Enterococcus faecium, we examined the antibiotic susceptibilities of clinical strains (n = 84) isolated at one institution during the 22 years since 1968. A significant increase in resistance to penicillin was observed during the study period: the MICs of penicillin for 50 and 90% of isolates tested were 16 and 64 micrograms/ml, respectively, from 1969 to 1988 (n = 48; geometric mean MIC, 14 micrograms/ml) , whereas they were 256 and 512 micrograms/ml, respectively, from 1989 to 1990 (n = 36; geometric mean MIC, 123 micrograms/ml) (P less than 0.001). A comparable increase in resistance to ampicillin was also noted (P less than 0.001). No strains produced detectable beta-lactamase. In contrast, susceptibilities to vancomycin, teicoplanin, and ciprofloxacin remained stable. High-level resistance to gentamicin was observed in none of 48 isolates from 1969 to 1988, but was present in 22 of 36 strains (61%) from 1989 to 1990 (P less than 0.001) and was significantly associated with resistance (MIC, greater than or equal to 128 micrograms/ml) to penicillin (P less than 0.001). To assess the potential evolution of antibiotic resistance in this species, clinical isolates (n = 24) were compared with strains isolated in 1968 from a human population in the Solomon Islands that was never exposed to antibiotics. Solomon Island isolates were significantly more susceptible than all clinical strains to penicillin, ampicillin, and vancomycin (P less than 0.001 for each), but they exhibited no differences in susceptibility to teicoplanin or ciprofloxacin. The penicillin-binding affinity of penicillin-binding protein 5 (PBP 5) in penicillin-resistant clinical strains (MIC, 512 micrograms/ml) was notably lower than that in strains with more typical susceptibilities, suggesting an alteration in this PBP as a possible mechanism for increased penicillin resistance. Solomon Island strains most susceptible to penicillin demonstrated a prominent PBP 5* and the absence of PBP 5. These changes in the antibiotic resistance of E. faecium emphasize the importance of identifying this species in patients with serious enterococcal infections and the necessity of assessing its susceptibility to both beta-lactams and aminoglycosides if effective therapy is to be identified.