The study of protein interactions constitutes an important domain to understand the physiology and pathogenesis of microorganisms. The two dimensional blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D BN/SDS-PAGE) was initially reported to analyze membrane protein complexes. In this study, both cytoplasmic and membrane complexes of a bacterium : the strain J99 of the gastric pathogen Helicobacter pylori, were analyzed by this proteomic approach. It was possible to identify 34 different proteins grouped in 13 multiprotein complexes: 11 from the cytoplasm and 2 from the membrane, either previously reported partially or totally in the literature. Beside complexes involved in H. pylori physiology, this method allowed the description of interactions involving known pathogenic factors such as i) urease with the heat shock protein GroEL or with the putative ketol-acid reductoisomerase IlvC, and ii) the cag pathogenicity island CagA protein with the DNA gyrase GyrA, as well as insight on the partners of TsaA, a peroxide reductase/stress dependent molecular chaperone. The 2D BN/SDS-PAGE combined with mass spectrometry is a potential tool to study the differences in complexes isolated in various situations and also to study the interactions between bacterial and eucaryotic cell proteins.