Metronidazole resistance in Helicobacter pylori has been attributed to mutations in rdxA or frxA. Insufficient data correlating RdxA and/or FrxA with the resistant phenotype, and the emergence of resistant strains with no mutations in either rdxA or frxA, indicated that the molecular basis of H. pylori resistance to metronidazole required further characterization. The rdxA and frxA genes of four matched pairs of metronidazole-susceptible and -resistant strains were sequenced. The resistant strains had mutations in either rdxA, frxA, neither gene, or both genes. The reduction rates of five substrates suggested that metabolic differences between susceptible and resistant strains cannot be explained only by mutations in rdxA and/or frxA. A more global approach to understanding the resistance phenotype was taken by employing two-dimensional gel electrophoresis combined with tandem mass spectrometry analyses to identify proteins differentially expressed by the matched pair of strains with no mutations in rdxA or frxA. Proteins involved in the oxireduction of ferredoxin were downregulated in the resistant strain. Other redox enzymes, such as thioredoxin reductase, alkyl hydroperoxide reductase, and superoxide dismutase, showed a pI change in the resistant strain. The data suggested that metronidazole resistance involved more complex metabolic changes than specific gene mutations, and they provided evidence of a role for the intracellular redox potential in the development of resistance.