Active efflux systems MexAB-OprM and MexXY were found to be overexpressed simultaneously in 12 multiresistant clinical isolates of Pseudomonas aeruginosa. Nine of these strains (agrZ mutants) harbored mutations in gene mexZ, the product of which down-regulates expression of operon mexXY. Eight of the 12 strains exhibited mutations in genes known to control transcription of operon mexAB-oprM, such as mexR (four nalB mutants) or PA3721 (three nalC mutants). 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 over-expression levels of genes mexA or mexX, and (iii) the resistance levels to effluxed antibiotics. Finally, three and four isolates overproduced MexXY (agrW mutants) or MexAB-OprM (nalD mutants), respectively, without any mutation in the known regulator genes. 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.