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Proton motive force (PMF) inhibition enhances the intracellular accumulation of autoinducers possibly interfering with biofilm formation. We evaluated the effect of the PMF inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP) on Pseudomonas aeruginosa biofilm development.Four epidemiologically unrelated P. aeruginosa isolates were studied. A MexAB-oprM overproducing strain was used as control. Expression of gene mexB was examined and biofilm formation after incubation with 0, 12·5 and 25 μmol l−1 of CCCP was investigated. Mean values of optical density were analysed with one-way analysis of variance and t-test. Two isolates subexpressed mexB gene and only 25 μmol l−1 of CCCP affected biofilm formation. Biofilms of the other two isolates and control strain PA140 exhibited significantly lower absorbance (P ranging from <0·01 to <0·05) with either 12·5 or 25 μmol l−1 of CCCP.The PMF inhibitor CCCP effect was correlated with the expression of MexAB-OprM efflux system and found to compromise biofilm formation in P. aeruginosa.These data suggest that inhibition of PMF-dependent trasporters might decrease biofilm formation in P. aeruginosa.