Escherichia coli was shown to have an energy-dependent reduced uptake of the fluoroquinolone antimicrobial agent norfloxacin. Studies of everted inner membrane vesicles suggested that this reduced accumulation involved a carrier-mediated norfloxacin active efflux generated by proton motive force with an apparent Km of 0.2 mM and a Vmax of 3 nmol min-1 mg of protein-1. Other hydrophilic, but not hydrophobic, quinolones competed with norfloxacin for transport. Porin (OmpF)-deficient E. coli cells were twofold less susceptible to norfloxacin and showed twice as much energy-dependent reduction in drug uptake. However, active efflux assayed in everted vesicles from the OmpF strain was unchanged compared with that in the parental strain. These findings suggest that in the OmpF mutant decreased outer membrane permeability, combined with active efflux across the inner membrane, in some manner results in decreased steady-state uptake of norfloxacin and lowered drug susceptibility.