The ability to colonize patients successfully may be essential for the emergence and spread of resistant nosocomial strains. We determined the presence of Esp, a surface protein involved in colonization ability in Enterococcus faecalis, in 96 Enterococcus faecium isolates from hospitalized patients (77 PFGE clones), 33 faecal isolates from healthy volunteers (32 clones) and 20 environmental isolates (20 clones). Esp was found significantly more often in E. faecium isolated from hospitalized patients than in isolates from the community setting (26% versus 6%, P < 0.01) and was significantly more common among ampicillin-resistant than among ampicillin-susceptible strains (37% versus 4%, P < 0.001), regardless of the isolation site. The frequency of the esp gene in the hospital clearly correlates with antibiotic-resistant E. faecium clones. This observation indicates that antibiotic-resistant variants may frequently arise under antibiotic selective pressure among esp-positive clones reaching ecological abundance in the nosocomial habitat.