The peptic ulcer-causing bacterium Helicobacter pylori was found to contain an H2-uptake hydrogenase activity coupled to whole cell (aerobic) respiration. The activity was localized to membranes which functioned in the H2-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 H2-oxidizing membrane-associated respiratory chain.