The 4-quinolone antibiotics nalidixic acid and ciprofloxacin are potent inhibitors of the bacterial type II topoisomerase DNA gyrase. Treatment of mouse L1210 leukemia cells with these drugs resulted in a delayed inhibition of cell proliferation. Prior to inhibition of cell proliferation, there was a time-dependent decrease in the cellular content of mitochondrial DNA (mtDNA). The decrease in mtDNA was associated with a decrease in the rate of mitochondrial respiration and an increase in the concentration of lactate in the growth medium. Inhibition of cell proliferation by 4-quinolones was reversible upon drug washout. However, there was a 2- to 4-day lag before the growth rate returned to normal levels. This was preceded by an increase in mtDNA content and mitochondrial respiration. These studies suggest that inhibition of mammalian cell proliferation by 4-quinolone drugs is related to the selective depletion of mtDNA.