To analyze the relationship between biomass of microorganisms and methane production, the total biomass of bacteria and archaea (BA) during methane fermentation was analyzed by the environmental DNA analysis method. In the case of using methanogenic sludge as a seed which is generally used for methane fermentation, the total BA biomass reached to 1.5 x 10(8) to 3.6 x 10(8) cells/ml when methane was produced. On the other hand, soil suspension was used as a seed; methane was not produced for 14-day cultivation. However, the total BA biomass reached to above 1.5 x 10(8) cells/ml. The methanogen biomass was counted by using a fluorescence microscope (coenzyme F420), and the methanogen biomass and the ratio of methanogens in the total of BA were analyzed during methane fermentation. At the methane-producing phase, the methanogen biomass reached to 1.3 x 10(8) cells/ml, and the ratio of methanogens was above 70% of the total BA. When the ratio of methanogens in a seed was changed, the methane-producing phase was moved. However, the relationship between methanogens and other microorganisms at the methane-producing phase was almost similar.