The MexA-MexB-OprM efflux pump of Pseudomonas aeruginosa consists of two inner membrane proteins, MexA and MexB, and one outer membrane protein, OprM. We investigated the role of the components of this drug extrusion system by evaluating the repercussions of deleting these subunit components on the accumulation of several fluorescent probes. Fluorescence intensities of positively charged 2-(4-dimethylaminostyryl)-1ethylpyridinium and uncharged N-phenyl-1-naphtylamine were 7 and 4 times higher, respectively, in the mutant lacking OprM and 4 and 1.7 times higher, respectively, in the mutants lacking MexA or MexB than in the wild type strain. This order of fluorescence intensity was fully consistent with a previously reported minimum inhibitory concentration of antibiotics such as tetracycline, chloramphenicol, and finoroquinolones. Ethidium bromide accumulation in all the Mex mutants proceeded at about 5 times faster than the rate in the wild type cells. This result is in accord with the minimum inhibitory concentration of β-lactam antibiotics. These results suggest that the fluorescence probes could be successfully used in real time monitoring of the function of the drug extrusion machinery in Gram-negative bacteria. The downhill extrusion kinetics of 1-(4trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, which orients perpendicular to the inner leaflet of the cytoplasmic membrane, from preloaded cells lacking the extrusion pump was preceded by a slow increase in fluorescence intensity, whereas the wild type cell immediately released the dye. This observation was explained by a slow transcytoplasmic membrane crossing of intracellular dye in the mutants. These results reflected higher accumulation of the probe in the cytoplasmic membrane in the mutants and strengthened the hypothesis that extrusion of hydrophobic substrate mediated by MexA-MexB-OprM mainly takes place from the interior of the cytoplasmic membrane.