The accumulation of sparfloxacin and three other fluoroquinolones of decreasing hydrophobicity, pefloxacin, ofloxacin, and ciprofloxacin, into randomly chosen fluoroquinolone-sensitive and -resistant clinical isolates of bacteria (four Enterobacteriaceae, one Pseudomonas aeruginosa and one Staphylococcus aureus) was measured. There was good correlation between the concentration of drug that inhibited early DNA synthesis in whole cells and the MIC values of the same drug regardless of organism sensitivity or the quinolone. A decrease in permeability in resistant strains compared with sensitive, due to a modification of outer membrane proteins was often involved in resistance. But, in all cases other mutations may also be involved explaining the relatively high level of resistance of the strains. In two resistant strains the DNA gyrase was purified and found to be resistant to inhibition by quinolones. The use of quinolones of differing hydrophobicity emphasized the importance of this property in the uptake of quinolones by bacterial cells, and provided evidence that sparfloxacin used both porin and the self-promoted uptake pathway for it's uptake.