To identify the methanogenic pathways present in a deep aquifer associated with an accretionary prism in Southwest Japan, a series of geochemical and microbiological studies of natural gas and groundwater derived from a deep aquifer were performed. Stable carbon isotopic analysis of methane in the natural gas and dissolved inorganic carbon (mainly bicarbonate) in groundwater suggested that the methane was derived from both thermogenic and biogenic processes. Archaeal 16S rRNA gene analysis revealed the dominance of H(2)-using methanogens in the groundwater. Furthermore, the high potential of methane production by H(2)-using methanogens was shown in enrichments using groundwater amended with H(2) and CO(2). Bacterial 16S rRNA gene analysis showed that fermentative bacteria inhabited the deep aquifer. Anaerobic incubations using groundwater amended with organic substrates and bromoethanesulfonate (a methanogen inhibitor) suggested a high potential of H(2) and CO(2) generation by fermentative bacteria. To confirm whether or not methane is produced by a syntrophic consortium of H(2)-producing fermentative bacteria and H(2)-using methanogens, anaerobic incubations using the groundwater amended with organic substrates were performed. Consequently, H(2) accumulation and rapid methane production were observed in these enrichments incubated at 55 and 65 degrees C. Thus, our results suggested that past and ongoing syntrophic biodegradation of organic compounds by H(2)-producing fermentative bacteria and H(2)-using methanogens, as well as a thermogenic reaction, contributes to the significant methane reserves in the deep aquifer associated with the accretionary prism in Southwest Japan. The ISME Journal (2010) 4, 531-541; doi:10.1038/ismej.2009.132; published online 3 December 2009