The contribution of the MexXY multidrug efflux system to aminoglycoside resistance was investigated in 18 clinical isolates of Pseudomonas aeruginosa obtained from dairy cows with Pseudomonas mastitis. All of the isolates expressed MexXY as determined by reverse transcription-PCR. The loss of mexXY resulted in increased susceptibility (two- to 16-fold decline in MIC) to aminoglycosides, confirming the contribution of this system in aminoglycoside resistance in these strains. As the impact of DeltamexXY varied, overexpression of MexXY alone is not sufficient for aminoglycoside resistance. Expression of mexXY also varied and did not strictly correlate with aminoglycoside insusceptibility. Transcription levels of mexY were independent on mutations in mexZ, suggesting the existence of additional regulatory mechanisms other than mexZ.