The peptic ulcer-causing bacterium Helicobacter pylori was found to contain an H 2 -uptake hydrogenase activity coupled to whole cell (aerobic) respiration. The activity was localized to membranes which functioned in the H 2 -oxidizing direction with a variety of artificial and physiological electron acceptors of positive redox potential. Immunoblotting of H. pylori membrane components with anti ( B. japonicum) hydrogenase large and small subunit-specific antisera identified H. pylori hydrogenase peptides of approximately 65 and 26 kDa respectively, and H. pylori genomic DNA fragments hybridizing to the ( B. japonicum) hydrogenase structural genes were identified. The membrane-bound activity was subject to anaerobic activation, like many NiFe hydrogenases. Difference absorption spectral studies revealed absorption peaks characteristic of b and c-type cytochromes, as well as of a bd-type terminal oxidase in the H. pylori H 2 -oxidizing membrane-associated respiratory chain.