Mutations in the gyrA gene resulting in amino acid changes at Ser-91 and Asp-95 are significantly associated with decreased susceptibilities to quinolones in Neisseria gonorrhoeae. To detect these mutations, we developed a rapid and simple assay based on amplification of the region of the gyrA gene containing the mutation sites by PCR and digestion of the PCR product with a restriction enzyme. A naturally occurring HinfI restriction site was present in the region containing the Ser-91 codon, and an artificial HinfI restriction site was created in the region containing the Asp-95 codon by the method of primer-specified restriction site modification. The mutations generating alterations at Ser-91 and Asp-95 were detected as restriction fragment length polymorphisms of the PCR products digested with HinfI. Fifty-five clinical strains of N. gonorrhoeae were examined for mutations in the gyrA gene by this method. Mutations at Ser-91 and/or Asp-95 were detected in all the 31 strains in which the mutations had been confirmed by DNA sequencing. Our method allows simultaneous testing of a large number of strains and provides results within 8 h. This rapid and simple assay could be a useful screening device for genetic alterations associated with decreased susceptibilities to quinolones in N. gonorrhoeae and could facilitate epidemiological studies on clinical isolates of N. gonorrhoeae with decreased susceptibilities to quinolones.