The MexAB-OprM efflux pump of Pseudomonas aeruginosa consists of two inner and one outer membrane proteins and exports xenobiotics rendering the cells resistant to structurally diverse antimicrobial agents. We used fluorescent probes to monitor the pump function. Fluorescence intensity of hydrophobic cation, 2-(4-dimethylaminostyryl)-1-ethylpyridinium, was 7 to 4 times higher in the mutant lacking the pump subunit than the wild type strain. Water-soluble fluorescent probe, ethidium bromide, accumulated in the Mex mutants at the rate 5 times faster than in the wild-type cells. The results demonstrated that the fluorescent probes are powerful tools in real-time monitoring the function of the efffux pump. Topological of the MexB subunit showed that the protein span the membrane 12 times and had two huge periplasmic domains. MexA is an inner membrane anchoring lipoprotein and exposing entire polypeptide domain to the periplasmic space. OprM anchored the outer membrane via fatty acids and exposed the protein portion to the periplasmic space. These results suggested that the MexAB-OprM pump exports xenobiotics as that MexB drafts the substrates at the cytoplasmic membrane and export to periplasmic space and MexA and OprM convey the substrate bypassing the periplasmic space to the outer membrane.