OBJECTIVE. To quantify the association between admission to an intensive care unit (ICU) room most recently occupied by a patient positive for extended-spectrum β-lactamase (EBSL)-producing gram-negative bacteria and acquisition of infection or colonization with that pathogen. DESIGN. Retrospective cohort study. SETTING AND PATIENTS. The study included patients admitted to medical and surgical ICUs of an academic medical center between September 1, 2001, and June 30, 2009. METHODS. Perianal surveillance cultures were obtained at admission to the ICU, weekly, and at discharge from the ICU. Patients were included if they had culture results that were negative for ESBL-producing gram-negative bacteria at ICU admission and had an ICU length of stay longer than 48 hours. Pulsed-field gel electrophoresis (PFGE) was performed on ESBL-positive isolates from patients who acquired the same bacterial species (eg, Klebsiella species or Escherichia coli) as the previous room occupant. RESULTS. Among 9,371 eligible admissions (7,651 unique patients), 267 (3%) involved patients who acquired an ESBL-producing pathogen in the ICU; of these patients, 32 (12%) were hospitalized in a room in which the prior occupant had been positive for ESBL. Logistic regression results suggested that the prior occupant's ESBL status was not significantly associated with acquisition of an ESBL-producing pathogen (adjusted odds ratio, 1.39 [95% confidence interval, 0.94-2.08]) after adjusting for colonization pressure and antibiotic exposure in the ICU. PFGE results suggested that 6 (18%) of 32 patients acquired a bacterial strain that was the same as or closely related to the strain obtained from the prior occupant. CONCLUSIONs. These data suggest that environmental contamination may not play a substantial role in the transmission of ESBL-producing pathogens among ICU patients. Intensifying environmental decontamination may be less effective than other interventions in preventing transmission of ESBL-producing pathogens.