To determine rates of colonization with multidrug-resistant (MDR) bacteria (ie, methicillin-resistant Staphylococcus aureus [MRSA], vancomycin-resistant Enterococcus [VRE], extended-spectrum beta -lactamase [ESBL]-producing Enterobacteriaceae, and Acinetobacter baumannii) after prolonged hospitalization and to assess the yield of surveillance cultures and variables associated with colonization with MDR bacteria. Prospective observational cohort study conducted from February 6 to May 26, 2006. All patients who spent more than 30 days in our university hospital (Paris, France) were included. Rectal and nasal swab samples obtained during day 30 screening were examined for MRSA, VRE, ESBL-producing Enterobacteriaceae, and A. baumannii. Of 470 eligible patients, 439 had surveillance culture samples available for analysis, including 51 patients (11.6%) with a history of colonization or infection due to 1 or more types of MDR bacteria (MRSA, recovered from 35 patients; ESBL-producing Enterobacteriaceae, from 16 patients; A. baumannii, from 6 patients; and VRE, from 0 patients) and 37 patients (9.5% of the 388 patients not known to have any of the 4 MDR bacteria before day 30 screening) newly identified as colonized by 1 or more MDR bacteria (MRSA, recovered from 20 patients; ESBL-producing Enterobacteriaceae, from 16 patients; A. baumannii, from 1 patient; and VRE, from 0 patients). A total of 87 (19.8%) of 439 patients were identified as colonized or infected with MDR bacteria at day 30. Factors that differed between patients with and without MRSA colonization included age, McCabe score, comorbidity score, receipt of surgery, and receipt of fluoroquinolone treatment. Patients with ESBL-producing Enterobacteriaceae colonization were younger than patients with MRSA colonization. Differences in the variables associated with MRSA colonization and ESBL-producing Enterobacteriaceae colonization suggest differences in the epidemiology of these 2 organisms. Day 30 screening resulted in a 72.5% increase in the number of patients identified as colonized with at least 1 type of MDR bacteria.