Cefepime (FEP) and ceftazidime (CAZ) are potent beta-lactam antibiotics with similar MICs (1 to 2 mug/ml) for wild-type strains of Pseudomonas aeruginosa. However, recent epidemiological studies have highlighted the occurrence of isolates more resistant to FEP than to CAZ (FEPr/CAZs profile). We thus investigated the mechanisms conferring such a phenotype in 38 clonally unrelated strains collected in two French teaching hospitals. Most of the bacteria (n=32; 84%) appeared to stably overexpress the mexY gene, which codes for the RND transporter of the multidrug efflux system MexXY-OprM. MexXY up-regulation was the sole FEP resistance mechanism identified (n=12) or was associated with increased levels of pump MexAB-OprM (n=5) or MexJK (n=2), synthesis of secondary beta-lactamase PSE-1 (n=10), derepression of cephalosporinase AmpC (n=1), coexpression of both OXA-35 and MexJK (n=1), or production of both PSE-1 and MexAB-OprM (n=1). Down-regulation of the mexXY operon in seven selected strains by the plasmid-borne repressor gene mexZ decreased FEP resistance from two- to eightfold, thereby demonstrating the significant contribution of MexXY-OprM to the FEPr/CAZs phenotype. The six isolates of this series that exhibited wild-type levels of the mexY gene were found to produce beta-lactamase PSE-1 (n=1), OXA-35 (n=4), or both PSE-1 and OXA-35 (n=1). Altogether, these data provide evidence that MexXY-OprM plays a major role in the development of FEP resistance among clinical strains of P. aeruginosa.