Polymyxins are often the only option to treat acquired multidrug-resistant Pseudomonas aeruginosa. Polymyxin susceptibility in P. aeruginosa PAO1 is associated with the lipopolysaccharide structure that is determined by arnBCADTEF and modulated by phoPQ and pmrAB. We examined five clonally unrelated clinical isolates of polymyxin B-resistant P. aeruginosa to investigate the molecular basis of polymyxin resistance. All isolates grew with 4 g/ml polymyxin B (MIC, 8 g/ml), whereas P. aeruginosa PAO1 grew with 0.25 g/ml polymyxin B (MIC, 0.5 g/ml). The resistant isolates were converted to susceptible ones (the MICs fell from 8 to 0.5 g/ml) following the introduction of phoPQ (four isolates) and pmrAB (one isolate), which had been cloned from strain PAO1. DNA sequence analysis revealed that a single-nucleotide substitution in three isolates replaced a single amino acid of PhoQ, the deletion of 17 nucleotides in one isolate truncated the protein of PhoQ, and two nucleotide substitutions in one isolate replaced two amino acids of PmrB. The involvement of these amino acid substitutions or the truncated protein of PhoQ and PmrB in polymyxin B resistance was confirmed using strain PAO1 lacking phoPQ or pmrAB that was transformed by phoPQ or pmrAB containing the amino acid substitutions or the truncated protein. The resistant clinical isolates were sensitized by the inactivation of arnBCADTEF (the MICs fell from 8 to 0.5 g/ml). These results suggest that polymyxin B resistance among clinical isolates of P. aeruginosa is associated with alterations in two-component regulatory systems of phoPQ or pmrAB.Pseudomonas aeruginosa is a nosocomial gram-negative opportunistic pathogen that causes a variety of infections (e.g., urinary tract, respiratory, skin, soft tissue, etc.) (3,12,18,19). P. aeruginosa accounts for 11 to 14% of all nosocomial infections and is a major problem for people hospitalized with cancer, cystic fibrosis, or burns (3). Treatment usually involves the use of one or more antibiotics, such as ␤-lactams, aminoglycosides, or quinolones. Combination therapy usually is recommended for P. aeruginosa infections, as it decreases the risk of antibiotic resistance and enhances the eradication rate. Despite the use of combination therapy, there are numerous reports of the emergence of multidrug-resistant P. aeruginosa. Polymyxins (polymyxin B and colistin) often have been the last resort to treat such isolates (1, 3, 27). However, polymyxin B resistance in multidrug-resistant clinical isolates has been reported (4, 6, 11, 25).The mode of action and the resistance mechanism to polymyxin B has been studied extensively using the reference P. aeruginosa strain PAO1. Polymyxin B is a polycationic lipopeptide antibiotic that interacts with a negatively charged lipid A moiety of the lipopolysaccharide (LPS) of gram-negative bacteria and leads to cell lysis and death (26). Resistance to polymyxin B is caused by the inhibition of the interactions between the antibiotic and the lipid A moiety of the LPS, and the inhibition is ba...