BACKGROUND AND OBJECTIVES: Multidrug-resistant gram-negative bacteria are rapidly spreading throughout the world. The epidemiology of multidrug-resistant gram-negative bacteria in patients who require chronic hemodialysis has not been previously studied. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A prospective cohort study of an outpatient hemodialysis unit was conducted. Serial surveillance cultures for multidrug-resistant gram-negative bacteria, vancomycin-resistant enterococci, and methicillin-resistant Staphylococcus aureus were collected from patients who were undergoing chronic hemodialysis. RESULTS: Nineteen (28%) of the 67 enrolled patients were colonized with one or more antimicrobial-resistant bacteria at study enrollment. Eleven (16%), nine (13%), and three (5%) patients were colonized with multidrug-resistant gram-negative bacteria, vancomycin-resistant enterococci, and methicillin-resistant Staphylococcus aureus, respectively. Independent risk factors associated with harboring multidrug-resistant gram-negative bacteria at enrollment were residence in a long-term care facility and antibiotic exposure for > or = 7 d in the previous 3 mo. Twenty-two (40%) of 55 patients who had follow-up cultures acquired at least one antimicrobial-resistant bacterium. A total of 20, 15, and 13% of patients acquired multidrug-resistant gram-negative bacteria, vancomycin-resistant enterococci, and methicillin-resistant Staphylococcus aureus, respectively. Antibiotic exposure was the only independent risk factor for multidrug-resistant gram-negative bacteria acquisition. Endogenous multidrug-resistant gram-negative bacteria acquisition was detected among 69% of acquired multidrug-resistant gram-negative bacterial strains. CONCLUSIONS: The prevalence and acquisition of multidrug-resistant gram-negative bacteria surpassed that of vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. Endogenous acquisition, as opposed to patient-to-patient spread, was the predominant mechanism of acquisition. Residence in a long-term care facility and antibiotic exposure may be important factors promoting the spread of multidrug-resistant gram-negative bacteria among this patient population.