Objectives: To determine the contribution to fluoroquinolone resistance of point mutations in the gyrA and parC genes of Salmonella Typhi. Methods: Point mutations that result in Ser-83?Phe, Ser-83?Tyr and Asp-87?Asn amino acid substitutions in GyrA and Glu-84?Lys in ParC were introduced into a quinolone-susceptible, attenuated strain of Salmonella Typhi using suicide vector technology. This is the first time that this approach has been used in Salmonella and abrogates the need for selection with quinolone antibacterials in the investigation of resistance mutations. Results: A panel of mutants was created using this methodology and tested for quinolone resistance. The ParC substitution alone made no difference to quinolone susceptibility. Any single GyrA substitution resulted in resistance to nalidixic acid (MIC = 512 mg/L) and increased by up to 23-fold the MIC of the fluoroquinolones ofloxacin (MIC = 2 mg/L) ciprofloxacin (MIC = 1 mg/L) and gatifloxacin (MIC = 0.38 mg/L). Among the double substitution mutants, those with a substitution in ParC were less prone to killing with ciprofloxacin. The triple substitution mutants (Ser-83?Phe or Tyr and Asp-87?Asn in GyrA with Glu-84?Lys in ParC) showed high levels of resistance to all the fluoroquinolones tested (MICs: gatifloxacin, 3–4 mg/L; ofloxacin, 32 mg/L; ciprofloxacin, 32–64 mg/L). Conclusions: In Salmonella Typhi the fluoroquinolones tested act on GyrA and, at higher concentrations, on ParC. The point mutations conferred reduced susceptibility to ofloxacin and ciprofloxacin, and also reduced susceptibility to gatifloxacin. Three mutations conferred resistance to ofloxacin (32 mg/L), ciprofloxacin (32 mg/L) and to the more active fluoroquinolone gatifloxacin (MIC = 3 mg/L). These results predict that the use of ofloxacin or ciprofloxacin will select for resistance to gatifloxacin in nature.