Fluoroquinolones are broad-spectrum antimicrobials highly effective in the treatment of a wide variety of clinical infections. Salmonella gastroenteritis is usually only treated with fluoroquinolones when the patient is elderly or immunocompromised. Fluoroquinolones are also used for the treatment of systemic Salmonella infection or for long-term salmonella carriage. Resistance to quinolones is commonly mediated by point mutations within the topoisomerase genes gyrA and parC. Pyrosequencing technology is a DNA sequencing method using 'sequencing by synthesis' and is suitable for the rapid detection of single nucleotide polymorphisms (SNPs). One hundred and ten Salmonella enterica isolates, representing 18 different serotypes, were used in this study. One hundred and four isolates had ciprofloxacin MICs of 0.25-32 microg/mL; the remaining six were ciprofloxacin-sensitive (ciprofloxacin MIC<or=0.125 microg/mL). PCR amplification of the quinolone resistance-determining regions of gyrA and parC was performed and the resulting amplicons subjected to a Pyrosequencing protocol using cyclic dispensation of nucleotides. Amino acid substitutions at S83 and D87 were detected in gyrA and at T57 and S80 in parC. Silent mutations were also identified at G81 of gyrA and at V67, H75 and H77 of parC. This is the first report of a Pyrosequencing assay being used to detect mutations in genes conferring quinolone resistance. The Pyrosequencing technology is a rapid and reliable alternative to current methods for identification of gyrA and parC mutations in S. enterica.