A prospective cohort study was undertaken to describe the epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBLKp) acquisition at an intensive care unit (ICU) in a non-outbreak setting. Surveillance for ESBLKp colonization and infection was performed in patients admitted at the ICU from January, 2000, to May, 2001. Screening for ESBLKp intestinal colonization was done by culturing rectal swab specimens at admission, 72 hr after admission and weekly until discharge or detection of ESBLKp. The incidence of ESBLKp intestinal colonization was 5.8/1,000 patient-days (95%CI, 3.4-10.1), and of ESBLKp infection was 1.7/1,000 patient-days (95%CI, 0.7-4.2). Use of vancomycin (OR 6.6; 95%CI, 1.73-25.28), amphotericin B (OR 12.0; 95%CI, 1.79-80.51), metronidazole (OR 5.3; 95%CI, 1.10-25.65), and ciprofloxacin (OR 0.1; 95%CI, 0.01-0.97) were independently associated with ESBLKp intestinal colonization. Previous ESBLKp colonization (OR 60.6; 95%CI, 56.33-578.73) was independently associated with ESBLKp infection. Each ICU-acquired ESBLKp isolate belonged to a different genotype by ERIC-PCR or pulsed-field gel electrophoresis (PFGE) and had a different plasmid profile, suggesting that cross transmission was not the main source for ESBLKp acquisition. Factors associated with ESBLKp in the non-outbreak setting were different from those previously reported during outbreaks. Intestinal ESBLKp colonization was confirmed as a risk factor for infection by this pathogen.