Transmission routes of Helicobacter pylori remain poorly understood. The finding of bacterial DNA in water suggests the involvement of environmental factors. Here we report successful co-cultivation of H. pylori with Acanthamoeba castellanii, which circumvents the requirement of this bacterium for precise microaerobic conditions and a large supply of nutrients in order to grow. H. pylori was able to propagate and remain viable for several weeks in the presence of amoebae under experimental conditions. Intact, metabolically active bacteria could be demonstrated in vacuoles. The putative dependence of H. pylori on free-living amoebae in nature could be important with respect to transmission and prevalence, as shown for some other pathogenic bacteria.