Simultaneous overexpression of the MexAB-OprM and MexXY efflux systems was demonstrated by real-time reverse transcription-PCR and immunoblotting experiments for 12 multiresistant clinical isolates of Pseudomonas aeruginosa. DNA sequencing analysis showed that nine of these strains (named agrZ mutants) harbored mutations in mexZ, the product of which downregulates the expression of the mexXY operon. In addition, 8 of the 12 strains exhibited mutations in genes known to control transcription of the mexAB-oprM operon. Four of them were nalB mutants with alterations in the repressor gene mexR, three of them appeared to be nalC mutants deficient in gene PA3721 and overexpressing gene PA3720, and one strain was a nalB nalC double mutant. For MexAB-OprM as well as for MexXY, no clear correlation could be established between (i) the types of mutations, (ii) the expression level of mexA or mexX, and (iii) resistance to effluxed antibiotics. Finally, three isolates, named agrW mutants, overproduced MexXY and had an intact mexZ gene, and four strains overproduced MexAB-OprM and had intact mexR and PA3721 genes (nalD mutants). These data show that clinical isolates are able to broaden their drug resistance profiles by coexpressing two Mex efflux pumps and suggest the existence of additional regulators for MexAB-OprM and MexXY.