Aims: To assess the different phenotypes and mechanisms of fluoroquinolone (FQ) resistance in clinical and environmental isolates of Escherichia coli. Methods and Results: We compared FQ-resistant E. coli isolates, measuring minimal inhibitory concentrations (MIC) of ciprofloxacin, along with susceptibility to other antibiotics. We also searched for the presence of efflux pumps, using efflux inhibitors, and for plasmid-borne FQ-resistance by PCR. We found that, aside from the higher FQ-resistance prevalence among clinical strains, environmental ones resist much lower concentrations of ciprofloxacin. Efflux pumps mediate fluoroquinolone resistance as frequently among environmental isolates than in clinical strains. Plasmid-borne qnrA genes were not detected in any resistant strain. Conclusions: Environmental FQ-resistant strains may have a nonclinical origin and/or a selective pressure different from the clinical use of FQs. Significance and Impact of the Study: The identification of the source of low-level FQ-resistant strains (ciprofloxacin MIC c. 8 mu g ml-1) in the environment could be important to curb the rapid emergence and spread of FQ-resistance in clinical settings, as these strains can easily become fully resistant to FQ concentrations achievable in fluids and tissues during therapy.