MexXY is an aminoglycoside-inducible multidrug transporter shown to contribute to intrinsic and acquired aminoglycoside resistance in laboratory isolates of Pseudomonas aeruginosa. To assess its contribution to aminoglycoside resistance in 14 clinical isolates demonstrating a panaminoglycoside resistance phenotype unlikely to be explained solely by aminoglycoside modification, expression of mexXY by these isolates was examined by reverse transcription-PCR. Elevated levels of mexXY expression were evident for most strains compared with those detected for an aminoglycoside-susceptible control strain, although there was no correlation between mexXY levels and the aminoglycoside MICs for the resistant strains, indicating that if MexXY was playing a role, other factors were also contributing. Deletion of mexXY from 9 of the 14 isolates resulted in enhanced susceptibilities to multiple aminoglycosides, confirming the contribution of this efflux system to the aminoglycoside resistance of these clinical isolates. Still, the impact of MexXY loss varied, with some strains clearly more or less dependent on MexXY for aminoglycoside resistance. Expression of mexXY also varied in these strains, with some showing high-level expression of the efflux genes independent of aminoglycoside exposure (aminoglycoside-independent hyperexpression) and others showing hyperexpression of the efflux genes that was to a greater or lesser degree aminoglycoside dependent. None of these strains carried mutations in mexZ, which encodes a negative regulator of mexXY expression, or in the mexZ-mexXY intergenic region. Thus, mexXY hyperexpression in aminoglycoside-resistant clinical isolates occurs via mutation in one or more as yet unidentified genes.