OBJECTIVE: To compare the effect of two strategies of antibiotic use (mixing vs. cycling) on the acquisition of resistant microorganisms, infections and other clinical outcomes. METHODS: Prospective cohort study in an 8-bed intensive care unit during 35- months in which a mixing-cycling policy of antipseudomonal beta-lactams (meropenem, ceftazidime/piperacillin-tazobactam) and fluoroquinolones was operative. Nasopharyngeal and rectal swabs and respiratory secretions were obtained within 48h of admission and thrice weekly thereafter. Target microorganisms included methicillin-resistant S. aureus, vancomycin-resistant enterococci, third-generation cephalosporin-resistant Enterobacteriaceae and non-fermenters. RESULTS: A total of 409 (42%) patients were included in mixing and 560 (58%) in cycling. Exposure to ceftazidime/piperacillin-tazobactam and fluoroquinolones was significantly higher in mixing while exposure to meropenem was higher in cycling, although overall use of antipseudomonals was not significantly different (37.5/100 patient-days vs. 38.1/100 patient-days). There was a barely higher acquisition rate of microorganisms during mixing, but this difference lost its significance when the cases due to an exogenous Burkholderia cepacia outbreak were excluded (19.3% vs. 15.4%, OR 0.8, CI 0.5-1.1). Acquisition of Pseudomonas aeruginosa resistant to the intervention antibiotics or with multiple-drug resistance was similar. There were no significant differences between mixing and cycling in the proportion of patients acquiring any infection (16.6% vs. 14.5%, OR 0.9, CI 0.6-1.2), any infection due to target microorganisms (5.9% vs. 5.2%, OR 0.9, CI 0.5-1.5), length of stay (median 5 d for both groups) or mortality (13.9 vs. 14.3%, OR 1.03, CI 0.7-1.3). CONCLUSIONS: A cycling strategy of antibiotic use with a 6-week cycle duration is similar to mixing in terms of acquisition of resistant microorganisms, infections, length of stay and mortality.