A cohort study was performed to investigate the risk factors for colonization with high-level aminoglycoside-resistant enterococci (HLARE) in intensive care unit (ICU) patients. Colonization was investigated by performing surveillance samples during ICU stay. Clonal relatedness of the isolates was assessed by pulsed-field gel electrophoresis. Eighty-six patients with an ICU stay of >48h were included; two were colonized with HLARE at admission, and 24 (28.5%) acquired HLARE during their stay in the ICU. HLARE were initially isolated from rectal swabs alone. Thirty-five percent of Enterococcus faecalis and 57% of E. faecium showed high-level resistance to gentamicin or streptomycin. Most isolates were clonally unrelated. Using multi-variate analysis, the only variable associated with HLARE colonization was previous antimicrobial use. Five patients had HLARE isolated from clinical samples, three of them with infection; in all of these, colonization with the same clone had been detected previously by surveillance samples. We conclude that most infections due to HLARE in the ICU are preceded by previous colonization, and that antimicrobial use is the main risk factor for colonization.