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To determine the impact of the rotation of antimicrobial agents on the rates of infection, intestinal colonization, and acquisition with antimicrobial-resistant Gram-negative bacteria.Pre- and postintervention design.A 19-bed, medical intensive care unit.Individuals admitted to the study unit for >48 hrs.After a 5-month baseline observation period, four classes of antimicrobial agents with Gram-negative activity were cycled at 3- to 4-month intervals for 24 months.The primary outcome was the acquisition rate of antimicrobial resistance among Enterobacteriaceae and Pseudomonas aeruginosa obtained from rectal swab cultures performed on admission, weekly during the patients’ stay, and at discharge. Rates and microbiology of nosocomial bloodstream infections and ventilator-associated pneumonia were also compared between baseline and cycling periods. The cycling program resulted in a significant change in prescribing practices; the predominant agent used changed with each cycle. Among study patients who were not already colonized with a resistant organism, the rate of acquisition of enteric colonization with bacteria resistant to any of the target drugs remained stable during the cycling period for P. aeruginosa (relative rate, 0.96; 95% confidence Interval, 0.47–2.16) and Enterobacteriaceae (relative rate, 1.57; 95% confidence interval, 0.80–3.43). Hospital-wide, P. aeruginosa from routine clinical cultures resistant to the target drugs increased during the cycling period. The proportion of Gram-negative bacteria isolated from cases of nosocomial bloodstream infection (29% baseline vs. 26% cycling; p = .11) and ventilator-associated pneumonia (80% vs. 41%; p = .06) did not significantly differ.In this study, antimicrobial cycling did not result in a significant change in enteric acquisition of resistant Gram-negative bacteria among intensive care unit patients.