Two mutations, one at the nalB and the other at the mexR locus, in the Pseudomonas aeruginosa chromosome are known to cause overexpression of the MexAB-OprM efflux pump. Based on the following results, we concluded that nalB is the mutation that has occurred within the mexR gene of the P. aeruginosa chromosome. (i) Nucleotide sequencing of the mex operon upstream region of 21 independent nalB-type mutants including the original nalB9 revealed that all the mutations were located within the mexR gene. The mutations were classified into three different groups and nine types including single base substitutions, single base deletions and base insertions. (ii) Substitution of the mutant mexR with the wild-type mexR and replacement of the wild-type mexR with a defined mexR mutation resulted in the expression of wild-type and nalB-type MexAB-OprM respectively, which was confirmed by testing the antibiotic susceptibility and beta-galactosidase activity of the mexA-lacZ translational fusion.