The administration of colistin is considered as the last alternative for infections by multidrug-resistant isolates of Pseudomonas aeruginosa; however its use is limited due to its considerable toxicity and poor pharmacokinetics. In order to define the in vitro activity of colistin combined with rifampin, 28 isolates resistant to piperacillin, ceftazidime, imipenem, meropenem, ciprofloxacin, amikacin, rifampin and colistin were tested. Seventeen of them that were found by PFGE to be genetically distinct were over time exposed to 2 microg/ml of colistin, to 2 microg/ml of rifampin and to their combination. Applied concentrations were selected to correspond to the mean serum level of the tested antimicrobials. Synergy between colistin and rifampin was found in four (23.5%), six (35.3%), seven (41.7%) and two (11.8%) isolates after 2, 4, 6 and 24 hours of growth, respectively. Bacterial re-growth was detected after 24 hours of exposure to the tested interaction. It is concluded that colistin and rifampin express a considerable in vitro synergistic effect on multidrug-resistant P. aeruginosa. The reported interaction should be tested in animal studies before introduction in clinical practice.