Two quinolone-susceptible Staphylococcus aureus and five quinolone-susceptible Streptococcus pneumoniae isolates were used to obtain in-vitro quinolone-resistant mutants in a multistep resistance selection process. The fluoroquinolones used were ciprofloxacin, moxifloxacin, levofloxacin, gemifloxacin, trovafloxacin and clinafloxacin. The mutagenicity of these quinolones was determined by the Salmonella and the Escherichia coli retromutation assays. All quinolone-resistant Staph. aureus mutants had at least one mutation in the grlA gene, while 86.6% of quinolone-resistant Strep. pneumoniae mutants had mutations in either or both the gyrA and parC genes. Moxifloxacin and levofloxacin selected resistant mutants later than the other quinolones, but this difference was more obvious in Staph. aureus. Accumulation of the fluoroquinolones by Staph. aureus did not explain these differences, since levofloxacin and moxifloxacin accumulated inside bacteria to the same extent as clinafloxacin and trovafloxacin. The results also showed that moxifloxacin and levofloxacin had less mutagenic potency in both mutagenicity assays, suggesting a possible relationship between the selection of resistance to quinolones and the mutagenic potency of the molecule. Furthermore, gemifloxacin selected efflux mutants more frequently than the other quinolones used. Thus, the risk of developing quinolone resistance may depend on the density of the microorganism at the infection site and the concentration of the fluoroquinolone, and also on the mutagenicity of the quinolone used, with moxifloxacin and levofloxacin being the least mutagenic. © 2005 Copyright by the European Society of Clinical Microbiology and Infectious Diseases.