Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged during recent years in several intensive care units. The objective of our study was to determine the incidence of CRKP and the risk factors associated with acquisition during intensive care unit (ICU) stay. This prospective cohort study was conducted between May 2007 and April 2008 in a medical-surgical ICU at a tertiary medical center. Rectal surveillance cultures were obtained from patients on admission and twice weekly. Of screened patients, 7.0% (21/299) were CRKP colonized on admission to the ICU. One hundred eighty (81%) patients were screened at least twice. Of these, 48 (27%) patients acquired CRKP during ICU stay. Of the 69 CRKP colonized patients (both imported and ICU acquired), 29% (20/69) were first identified by microbiologic cultures, while screening cultures identified 49 patients (71%). Of these, 23 (47%) subsequently developed clinical microbiological cultures. Independent risk factors for CRKP acquisition included recent surgery (OR 7.74; CI 3.42-17.45) and SOFA score on admission (OR 1.17; CI 1-1.22). In conclusion, active surveillance cultures detected a sizable proportion of CRKP colonized patients that were not identified by clinical cultures. Recent surgical procedures and patient severity were independently associated with CRKP acquisition.