The epidemiology of patients associated with ampicillin-resistant Enterococcus faecium (ARE) was investigated by combining both clinical approach and molecular analysis in a kidney transplant patient's ward. A case-control study was performed to identify risk factors for ARE by matching each patient with ARE with two control patients without any isolated E. faecium strain. ARE isolates were characterized by pulsed-field gel electrophoresis. From June 2004 to May 2006, 18 cases with clinical ARE samples were detected and compared with 35 control patients. By univariate analysis, recurrent urinary tract infections (UTIs) (odds ratio [OR], 4.9; 95% confidence interval [CI], 1.0-25.6), mean number of hospitalization days in the last year (p < 0.003), pyelonephritis or UTI (OR, 9.6; 95% CI, 2.2-46.1), oral third-generation cephalosporin use (OR, 12.42; 95% CI, 2.04-109.1), and fluoroquinolone use (OR, 4.4; 95% CI, 1.1-18.2) were significantly associated with ARE urinary tract colonization. By conditional logistic regression, hospitalization >21 days within 1 year (adjusted OR [aOR], 6.9; 95% CI, 1.0-46.5), recent medical history of pyelonephritis or UTI (aOR, 8.6; 95% CI, 1.5-49.1), and prior oral third-generation cephalosporin use (aOR, 13.1; 95% CI, 1.2-142.6) were identified as independent factors associated with ARE urinary tract colonization. Genotyping revealed a heterogeneous epidemiological situation with two major clones in patients hospitalized in successive rooms and 10 different single pulsotypes. Emergence of highly resistant enterococcal strains is a collateral damage from antibiotic prescription and represents a potential source of patient-to-patient transmission. Combining epidemiological approach and molecular analysis is a powerful tool to delineate mechanisms of emerging resistance. Improving our knowledge on ARE emergence in high antibiotic pressure hospital wards is a key factor to better control these colonizations/infections and to prevent the emergence of vancomycin-resistant E. faecium.