In 10 clinical isolates of Escherichia coli the frequency of spontaneous mutation to high levels of resistance to nalidixic acid was >300-fold higher than that to norfloxacin. Norfloxacin resistant mutants could not be detected (mutation frequency: <10⁻¹²). However, nalidixic acid resistant mutants of E. coli developed decreased susceptibility to norfloxacin at a rate of approximately 10⁻¹⁰. Mutants with decreased susceptibility to norfloxacin could also be obtained when the same clinical isolates of E. coli were exposed to norfloxacin in 2 steps. However, mutants with MIC values >8 μg/ml for norfloxacin were never obtained even after repeated exposure. Widespread use of cinoxacin or nalidixic acid may select resistant strains and reduce the efficacy of norfloxacin and other 4-quinolones.