In this study, the resistance mechanism of Pseudomonas aeruginosa to HK3140 (Fig. 1)-a new fluoroquinole-was characterized using a HK140 susceptible strain and a HK3140 strain which was selected among clinically isolated ofloxacin resistant P. aeruginosa strains during ofloxacin therapy. The susceptible and the resistant strains showed a remarkable difference in their outer membrane protein profiles. HK3140 concentrations inside the susceptible and the resistant strains were increased in the presence of carbonylcyanide m-chlorophenyl hydrazone (CCCP). HK3140 concentrations inside the resistant strain was increased in the presence of KNO₃, too. These results were confirmed by measuring the in uiuo DNA synthesis in the presence of CCCP and KNO₃, CCCP decreased the in uiuo DNA synthesis in both strains while KNO₃ decreased DNA synthesis only in the resistant strain. When DNA synthesis in the permeabilized cells were measured in the presence of HK3140, the amount of DNA synthesized in the permeabilized resistant strain was not greater a than that in the susceptible strain. Results suggested that the resistance of P. aeruginosa to HK3140 is a result from a change in permeability from changes in outer membrane proteins and an occurrence of a new ATPase dependent efflux system in addition to an intrinsic proton gradient dependent efflux system, but not from a change in the DNA gyrase.