An unusual compound, 1,3,4,6-hexanetetracarboxylic acid, was identified by H and C two-dimensional nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as one of the major components of the small-molecule pool in Methanobacterium thermoautotrophicum DeltaH under optimal conditions of cell growth. Incorporation of C- and H-labeled acetates was consistent with the biosynthesis of this tetracarboxylic acid from alpha-ketoglutarate, two molecules of acetyl-coenzyme A, and one molecule of CO(2), as established for the tetracarboxylic acid moiety of methanofuran. CO(2) pulse- CO(2) chase methodology was used to establish the turnover rate for this compound. In contrast to the two other major solutes in this bacterium, cyclic 2,3-diphosphoglycerate and glutamate, which are key metabolic intermediates, this free tetracarboxylic acid was metabolically inactive, with a half-life that exceeded the cell doubling time. Hence, this molecular pool cannot serve as a metabolic intermediate in cell biosynthesis. The functional role of free tetracarboxylate as a conservative part of a system that maintains high positive internal osmotic pressure in this bacterium is proposed.