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Antibiotic susceptibility methods that are commonly used to test bacterial isolates from patients with cystic fibrosis are of uncertain reliability for the polymyxins. To assess the reliability of four standard testing methods, this pilot study used a challenge set that included polymyxin-resistant isolates of Pseudomonas aeruginosa and Stenotrophomonas maltophilia.Twenty-five P. aeruginosa and 12 S. maltophilia isolates were tested for susceptibility to colistin (polymyxin E). Repeatability (concordance of replicates performed concurrently), reproducibility (concordance of replicates performed over time) and comparability (concordance of different methods) of agar dilution, broth microdilution, Etest and disc diffusion were assessed through the use of descriptive statistics and scatterplot analyses.All four methods displayed excellent repeatability (overall concordance rate of 99%). However, analysis of reproducibility revealed substantially lower rates of concordance (74% for agar dilution, 84% for broth microdilution and Etest, and 91% for disc diffusion). In addition, comparability to agar dilution of the three other methods was generally poor, with overall rates of very major error ranging from 12% for broth microdilution to 18% for Etest and disc diffusion.Compared with agar dilution, other susceptibility testing methods give high rates of apparent false polymyxin susceptibility for cystic fibrosis isolates of P. aeruginosa and S. maltophilia. Prospective study of the correlation between in vitro susceptibility and clinical response is needed to clarify whether these discrepancies reflect oversensitivity of the agar dilution method or insensitivity of the other methods.