Fluoroquinolones and carbapenems are important drug classes used for the treatment of Pseudomonas aeruginosa infections. However, overexpression of the P. aeruginosa efflux pump, MexEF-OprN, can provide dual resistance to both fluoroquinolones and carbapenems. Recently, a hospital in Texas encountered several P. aeruginosa isolates resistant to both of these drug classes. The purpose of this study was to determine whether the mechanism responsible for this multidrug resistance involved the overexpression of MexEF-OprN. To test this hypothesis, 7 clinical isolates from the Texas hospital were analyzed for clonality, antimicrobial susceptibility, expression of the porin, oprD, and four multidrug-resistant efflux pumps (mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY), quinolone resistance-determining region mutations, and beta-lactamase production. Two groups of isolates possessed similar pulse field gel electrophoresis patterns indicating their genetic relatedness. In addition to fluoroquinolone and carbapenem resistance, each isolate also displayed varying degrees of susceptibility to additional beta-lactams tested and resistance to gentamicin. Interestingly, none of the 7 clinical isolates overexpressed mexEF-oprN as determined by semiquantitative reverse transcriptase polymerase chain reaction, but overexpression of mexXY was observed in 6 of the 7 isolates. All 7 isolates showed a decrease of OprD in the outer membrane and a reduction in transcriptional expression of oprD compared to wild-type strain, PAO1. These results demonstrate that multidrug resistance to the fluoroquinolones and carbapenems in these clinical isolates was not a result of the overexpression of the mexEF-oprN pump. Instead, resistance to these two agents seemed to arise through independent mutational events.