We report the successful cultivation and partial characterization of novel members of epsilon-Proteobacteria, which have long been recognized solely as genetic signatures of small subunit ribosomal RNA genes (rDNA) from a variety of habitats occurring in deep-sea hydrothermal fields. A newly designed microhabitat designated 'in situ colonization system' was used for enrichment. Based on phylogenetic analysis of the rDNA of the isolates, most of these represent the first cultivated members harboring previously uncultivated phylotypes classified into the Uncultivated epsilon-Proteobacteria Groups A, B, F and G, as well as some novel members of Group D. Preliminary characterization of the isolates indicates that all are mesophilic or thermophilic chemolithoautotrophs using H(2) or reduced sulfur compounds (elemental sulfur or thiosulfate) as an electron donor and O(2), nitrate or elemental sulfur as an electron acceptor. The successful cultivation will enable the subsequent characterization of physiological properties and ecological impacts of a diversity of epsilon-Proteobacteria in the deep-sea hydrothermal environments.