BACKGROUND Ciprofloxacin (500 mg orally, single dose) is one of the recommended therapies for gonorrhea in Canada. In Canada, the first ciprofloxacin-resistant (CipR) Neisseria gonorrhoeae strain was isolated in 1993. Antimicrobial susceptibilities of N gonorrhoeae isolates were monitored as part of a national surveillance program to ensure efficacy of antimicrobial therapies. OBJECTIVE The goal was to determine the characteristics of ciprofloxacin resistance in Canadian gonococcal isolates. METHODS Susceptibility testing was performed on gonococcal strains from different provinces in Canada to determine the prevalence of CipR strains and their distribution. The CipR strains were further differentiated according to auxotype (A), serotype (S), plasmid profile (P), and pulsed-field gel electrophoresis (PFGE) profile. DNA sequencing and DNA microarray technology were used to determine mutations in gyrA and parC. RESULTS In Canada, between 1997 and 1999, 4.8% of resistant strains (130 of 2687 antibiotic-resistant N gonorrhoeae isolates) were CipR (MICs of 1-32 microg/l) and belonged to 48 A/S/P classes. Sixty-eight of the strains that were not differentiated by A/S/P were subtyped into 47 classes with PFGE. DNA sequencing and DNA microarray showed that the most common mutations had amino acid substitutions of Ser-->Phe at codon 91 and Asp-->Gly at codon 95 of the gyrA and Ser-->Arg at codon 87 of parC. CONCLUSIONS The CipR strains isolated in Canada are phenotypically and genotypically diverse, indicating that they were imported from overseas and not endemic in Canada. Mutations in gyrA and parC previously only identified by DNA sequencing were successfully identified with DNA microarray technology. DNA microarray technology could be an alternative tool for identifying point mutations in resistance genes or other epidemiologic markers when clinical laboratories replace culture methods with rapid and automated molecular methods for diagnosis.