A real-time PCR assay was developed to identify ciprofloxacin-resistant Campylobacter jejuni. Ciprofloxacin resistance in C. jejuni has been associated with a C-->T nucleotide point mutation occurring at the 86 codon of the gyrA gene. Other nucleotide substitutions have been identified in proximity to or at the same codon in the gyrA gene, but their role in ciprofloxacin resistance is still unknown. The LightCycler assay is based on the fluorescence resonance energy transfer technology using melting peak analysis of two fluorescent probes hybridized on PCR amplicons. This assay was used to detect the 86-codon mutation conferring ciprofloxacin resistance, as well as other nucleotides substitutions occurring within the same site in the gyrA gene. This gyrA mutation assay allows a rapid and reproducible screening method of ciprofloxacin resistant strains and was applied to C. jejuni strains isolated in Italy in 2000.