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Accretionary prisms are thick masses of sedimentary material scraped from the oceanic crust and piled up at convergent plate boundaries found across large regions of the world. Large amounts of anoxic groundwater and natural gas, mainly methane (CH4), are contained in deep aquifers associated with these accretionary prisms. To identify the subsurface environments and potential for CH4 production by the microbial communities in deep aquifers, we performed chemical and microbiological assays on groundwater and natural gas derived from deep aquifers associated with an accretionary prism and its overlying sedimentary layers. Physicochemical analyses of groundwater and natural gas suggested wide variations in the features of the six deep aquifers tested. On the other hand, a stable carbon isotope analysis of dissolved inorganic carbon in the groundwater and CH4 in the natural gas showed that the deep aquifers contained CH4 of biogenic or mixed biogenic and thermogenic origins. Live/dead staining of microbial cells contained in the groundwater revealed that the cell density of live microbial cells was in the order of 104 to 106‍ ‍cells‍ ‍mL-1, and cell viability ranged between 7.5 and 38.9%. A DNA analysis and anoxic culture of microorganisms in the groundwater suggested a high potential for CH4 production by a syntrophic consortium of hydrogen (H2)-producing fermentative bacteria and H2-utilizing methanogenic archaea. These results suggest that the biodegradation of organic matter in ancient sediments contributes to CH4 production in the deep aquifers associated with this accretionary prism as well as its overlying sedimentary layers.