The effects of fleroxacin and other new 4-quinolones on cellular functions and topoisomerase II of HeLa cells were investigated. The potency of cytotoxicity increased in the order fleroxacin < nalidixic acid < ofloxacin < norfloxacin < ciprofloxacin. The cytotoxicity was well correlated with inhibitory effects of 4-quinolones on topoisomerase II. However, the cytotoxicity did not correlate with antibacterial activity, and cytotoxicity was observed only at greater than 100-fold higher concentrations than the minimum inhibitory concentrations of new 4-quinolones for Enterobacteriaceae. Judging from the serum levels attainable after oral administration, new 4-quinolones, especially fleroxacin, will have little effect on topoisomerase in eukaryotic cells.