In Denmark surveillance of the in vitro susceptibility to ciprofloxacin of Neisseria gonorrhoeae was established in 1990. The proportion of N. gonorrhoeae strains with decreased susceptibility or resistance to ciprofloxacin (MIC >/= 0.06 microg/ml) was low (0.3 to 2.3%) up to 1995. Between 1995 and 1998 the rate of less-susceptible and resistant strains rose from 6.9 to 13.2%. Among ciprofloxacin-resistant strains (MIC >/= 1 microg/ml), 81% were highly resistant (MIC >/= 4 microg/ml). Thirty-five N. gonorrhoeae strains (40 isolates) for which ciprofloxacin MICs were 4 to 32 microg/ml were investigated for the frequency and patterns of mutations within the gyrA and parC genes. The quinolone resistance-determining regions of the gyrA and parC genes were amplified by PCR, and the amplicons were directly sequenced. Alterations at Ser-91 and Asp-95 in GyrA and a single or double alteration in ParC were identified in 32 strains (91%). Ser-91-to-Phe and Asp-95-to-Gly alterations in GyrA were detected in 28 strains (80%). The most common ParC alteration, Asp-86 to Asn, was found in 19 strains (54%). The strains were analyzed for genetic relationship by pulsed-field gel electrophoresis (PFGE). The analysis showed that nine strains with the same mutation pattern in the gyrA and parC genes, originating from different geographical areas over 3 years, had the same PFGE patterns after SpeI as well as NheI digestion (only one strain with one band difference in the NheI pattern), suggesting that a resistant clone had spread worldwide. The results from this study strongly suggest that double gyrA mutations plus a parC mutation(s) play an important role in the development of high-level fluoroquinolone resistance in N. gonorrhoeae.