In the course of an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) in an intensive care unit (ICU), we conducted active surveillance to determine the risk factors for ESBL-KP faecal colonization of patients. We used weekly rectal samples during a four-month period. ESBL-KP was found in the faeces of 72 of 188 (38%) patients, and 42 (58%) of them were colonized within the first week of admission to the ICU. The probability of remaining free of faecal colonization was less than 20% at 30 days of ICU admission. The risk factors associated with ESBL-KP faecal colonization were clinical severity score at admission (P = 0.004), arterial catheterization (P = 0.002), total parenteral nutrition (P = 0.04), urinary catheterization (P = 0.01), mechanical ventilation (P < 0.001), and previous antibiotic therapy (P = 0.04). A logistic regression analysis identified duration of urinary catheterization (OR:3.5; 95% CI 1.2-10.3) and mechanical ventilation (OR:4.6; 95% CI 1.1-19.3) as independent risk factors for ESBL-KP faecal colonization. Our results suggest that in an ESBL-KP prevalent environment, manipulations that facilitate cross-infection are the most relevant in the acquisition of the micro-organism and risk increases throughout hospitalization.