Quinolones are antimicrobial agents that act by inhibiting the DNA-gyrase and topoisomerase IV. The resistance to this family of antimicrobial agents has been steadily rising in recent years. The mechanisms of quinolone resistance are mutations in both targets and/or membrane alterations. The most important mutation causing quinolone resistance is in the codon for Ser-83 of the DNA-gyrase. This mutation affects a HinfI restriction point. This study was carried out to analyze the reliability of the PCR and RFLP with HinfI to study quinolone-resistance. A total of 200 clinical isolates were analyzed. All (159 clinical isolates) nalidixic acid resistant strains (MIC 128 μg/ml) showed the disappearance of the Ser-83 HinfI restriction point, whereas in the 41 clinical isolates with nalidixic acid MIC 4 μg/ml this restriction point was present. Six nalidixic acid susceptible strains (MIC 4 μg/ml) that showed a ciprofloxacin MIC of 0.125-0.25 did not present any alteration, whereas 22 clinical isolates with the same range of ciprofloxacin MIC but resistant to nalidixic acid with an MIC of 128-256 μg/ml, showed the disappearance of the HinfI restriction point correspondent to Ser-83. These results showed that RFLP with HinfI is a good method to perform rapid quinolone resistance screening.