Of the six putative small multidrug resistance (SMR) family proteins of Pseudomonas aeruginosa, a protein encoded by the PA4990 gene (emrE(Pae)) shows the highest identity to the well-characterized EmrE efflux transporter of Escherichia coli. Reverse transcription-PCR confirmed the expression of emrE(Pae) in the wild-type strain of P. aeruginosa. Using isogenic emrE(Pae), mexAB-oprM, and/or mexB deletion mutants, the contributions of the EmrE protein and the MexAB-OprM efflux system to drug resistance in P. aeruginosa were assessed by a drug susceptibility test carried out in a low-ionic-strength medium, Difco nutrient broth. We found that EmrE(Pae) contributed to intrinsic resistance not only to ethidium bromide and acriflavine but also to aminoglycosides. In this low-ionic-strength medium, MexAB-OprM was also shown to contribute to aminoglycoside resistance, presumably via active efflux. Aminoglycoside resistance caused by these two pumps could not be demonstrated in high-ionic-strength media, such as Luria broth or Mueller-Hinton broth. The EmrE-dependent efflux of ethidium bromide was confirmed by a continuous fluorescence assay.