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The objective of this study was to use immunoblotting with RdxA antisera to examine the production of the RdxA protein in mouse-derived metronidazole-susceptible and -resistant isolates of Helicobacter pylori. A 24 kDa immunoreactive band corresponding to RdxA was observed in all 15 metronidazole-susceptible and five of 50 metronidazole-resistant isolates. The rdxA gene of these five isolates contained missense mutations and transformation experiments confirmed that these mutations were associated with inactivation of the rdxA gene. No RdxA protein was produced in the other 45 metronidazole-resistant strains, including one in which the nucleotide sequence of the rdxA gene was unchanged. These results demonstrate a high correlation between production of the RdxA protein and susceptibility of H. pylori to metronidazole. Testing for the absence of the RdxA protein identifies the majority of strains that will respond poorly to metronidazole-containing eradication regimens.