The accumulation of quinolones by Escherichia coli JF568, Pseudomonas aeruginosa PAO1, and Staphylococcus aureus ATCC 29213 was measured by a modified fluorometric assay (J. S. Chapman and N. H. Georgopapadakou, Antimicrob. Agents Chemother. 33:27-29, 1989). The quinolones examined were fleroxacin, pefloxacin, norfloxacin, difloxacin, A56620, ciprofloxacin, ofloxacin, and Ro 09-1168. In all three organisms, uptake was complete in less than 5 min and was proportional to extracellular quinolone concentrations between 2 and 50 ;ag/ml, which is consistent with simple diffusion. Washing cells with quinolone-free buffer decreased accumulation by up Fluoroquinolones are synthetic antibacterial agents with marked bactericidal activities, broad antibacterial spectra, and favorable pharmacokinetics after oral or parenteral administration (25,36,44,48). Their molecular target is DNA gyrase, a unique and essential bacterial enzyme that catalyzes the negative supercoiling (unwinding) of doublestranded DNA and the decatenation of chromosomes after replication (15, 17-19, 23, 24). Quinolones thus interfere with a variety of processes involving DNA, such as replication, chromosomal segregation, transcription, and recombination.The ability of quinolones to enter bacterial cells is a factor contributing to their antibacterial potency (4, 41). Both the outer and cytoplasmic membranes are involved; alterations in either or both are associated with decreased quinolone accumulation and low-level resistance (2, 5-7, 10, 43, 47). In enterobacteria, quinolones diffuse across the outer membrane through the porin channels (13,21,26,27,37) and the phospholipid layer after disrupting the lipopolysaccharide (LPS) (8,20). In Pseudomonas aeruginosa, they diffuse through the D2 protein in the outer membrane and probably through the phospholipid bilayer similarly to enterobacteria (12,16,22,31,33,49). The cytoplasmic membrane is less of a barrier for quinolones, which enter by simple diffusion (2, 5, 26). However, quinolone accumulation is reduced by an active efflux system which is especially prominent in quinolone-resistant strains (11, 29, 51). Nevertheless, permeability plays a secondary role in quinolone susceptibility and resistance, in sharp contrast to the situation with ,B-lactam antibiotics (37).In this study, we have examined the accumulation of several quinolones in Escherichia coli, P. aeruginosa, and Staphylococcus aureus by using a modification of a previously described fluorometric assay (9