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Two target sites in Japan Sea, Toyama Bay and Shiribeshi Seamount, were studied using deep-sea submersibles to 'dowse' or imply seafloor methane seepage by analysis of ester-linked phospholipid fatty acids in sediments. No methane seeps in the studied sites had been reported before. The sediments associated with yellow to red patches showed a relatively high abundance of phospholipid fatty acids with biomarkers for sulfate-reducing bacteria and methane-oxidizing bacteria. Similar phospholipid fatty acid compositions were found in the sediments near the dense colonies of the deep-sea tunicate Megalodicopia hians . The tunicate colonies were first discovered in the coastal Japan area by this study, and their association with possible methane seepage was suspected. These results suggest that microflora with sulfate-reducing and methane-oxidizing bacteria may have developed in adaptation to methane seepage in the bathyal Japan Sea floor, where gas hydrate is likely formed beneath. In addition, supplement 16S rDNA data from the yellow patch sediment in Toyama Bay suggested that sulfur-oxidizing bacteria occur in response to sulfide supplied by sulfate-reducing bacteria. As methane is a more effective greenhouse gas than carbon dioxide and thus has more impact on global biosphere from aquatic systems, these observations call for the survey of the distribution of methane seeps associated with faults and subsurface gas hydrate in the coastal Japan Sea. [ABSTRACT FROM AUTHOR]