To investigate the mechanism of activation of the genes for resistance-nodulation-division (RND) family members MexE, MexF, and OprN for multidrug resistance (MDR), we mutagenized aefR and mexT, the potential regulators of mexEF/oprN transcription in Pseudomonas syringae pv. tabaci 6605 (Pta 6605). AefR is a member of the TetR transcription factors, and is known to be required for production of the quorum-sensing molecules, acyl homoserine lactones (AHL), in P. syringae. Furthermore, we found that AHL-synthesis-defective mutant strains in Pta 6605 showed enhanced expression of mexEF/oprN, and were highly tolerant to antimicrobial compounds such as chloramphenicol. MexT is a LysR-type transcription factor and is known to positively regulate transcription of mexEF/oprN in Pseudomonas aeruginosa. The ∆aefR mutant reduced the amount of growth in in vitro culture, caused the loss of AHL production, reduced the swarming motility, virulence and expression of psyI (AHL synthase) and psyR (AHL transcriptional regulator), and enhanced mexEF/oprN expression and tolerance to chloramphenicol, whereas the ∆mexT mutant retained the ability to produce AHL and did not show remarkable changes in in vitro growth, tolerance to antimicrobial compounds or virulence. Furthermore, unlike P. aeruginosa, the expression of mexEF/oprN is independent of MexT. These results indicate that (1) AefR is a regulator for the quorum-sensing system and MDR, and is required for swarming motility and virulence toward the host tobacco plant, and (2) MexT is not involved in the expression of mexEF/oprN in this bacterium.