Two patients with acute leukemia developed Escherichia coli bacteremia while receiving oral ofloxacin for antibacterial prophylaxis during profound neutropenia. The isolates were resistant to ofloxacin (MIC 25 and 12.5 micrograms/ml respectively), other fluoroquinolones and several unrelated agents. Whole cell drug accumulation studies with four different fluoroquinolones suggested major differences between drugs but only minor alterations in individual drug permeability in the two resistant isolates compared with a susceptible control strain. In a supercoiling assay using the purified DNA gyrase and plasmid pBR322, high concentrations (> or = 250 micrograms/ml) of both ofloxacin and ciprofloxacin were needed for visible inhibition of enzyme activity suggesting mutations in the DNA gyrase gene as the significant mechanism of resistance in both strains.