Fluoroquinolones are potent antibacterial agents that are being used as therapeutic agents for the treatment of multidrug-resistant tuberculosis. To better understand fluoroquinolone action in mycobacteria, the effects of ciprofloxacin were examined. DNA synthesis was inhibited rapidly in Mycobacterium smegmatis, DNA cleavage was readily observed by an empirical assay of cell lysate viscosity, and cell growth was blocked. These data are explained by the formation of gyrase-DNA-ciprofloxacin complexes that block replication fork movement. The bactericidal action of ciprofloxacin against M. smegmatis, Mycobacterium bovis BCG, and Escherichia coli occurred more slowly in cells with longer doubling times. The bactericidal effect against M. bovis BCG was partially blocked by pretreatment with chloramphenicol, an inhibitor of protein synthesis, and by very high concentrations of ciprofloxacin itself. Similar responses occur when E. coli is treated with ciprofloxacin. These similarities between E. coli and mycobacteria indicate that results from extensive fluoroquinolone studies with E. coli can be applied to mycobacteria. A simple viscometric assay of DNA cleavage is described. The assay is expected to be useful for screening new fluoroquinolone derivatives for increased effectiveness against clinically important bacteria.