The reliability of the Epsilometer-test (E-Test) and the disk diffusion (DD) method in the assessment of susceptibility of Helicobacter pylori (H. pylori) to metronidazole has recently been questioned, with possible clinical implications for the management of patients undergoing H. pylori eradication. The aims of this study were: 1) to compare the E-Test and disk diffusion methods to the agar dilution method for determining the susceptibility of H. pylori to metronidazole; and 2) to investigate whether potential discrepancies could be caused by the simultaneous presence of metronidazole susceptible and metronidazole resistant bacterial subpopulations.