The metabolic origin of formate, a substrate for methanogenesis in the rumen, was investigated by in vitro culture experiments with mixed ruminal microbes harvested from a goat. Since formate was rapidly metabolized to methane, formate was not detected in cultures of a usual mixed-microbial population. Therefore, methanogenesis was completely suppressed with anthraquinon, a methanogen inhibitor. This condition brought about the accumulation of H₂, which in turn caused the accumulation of formate by suppression of the reaction from formate to H2. Analysis on the stoichiometric relationship between substrates and products revieled that formate is produced from organic acids that are related with the glyoxylate cycle, such as citrate, isocitrate, 2-oxoglutarate, glyoxylate, and oxalate, by a pathway other than the pyruvate formate lyase reaction, which is the major route of formate production in the usual rumen. Especially formate production from oxalate may be important, because plants sometimes contain a large amount of oxalate.