A unique cyclic pyrophosphate compound has been detected at 10-12 mM intracellular concentration in Methanobacterium thermoautotrophicum by in vivo(31)P NMR. This compound has been extracted from cells and purified by anion-exchange chromatography. Studies with (1)H, (13)C, and (31)P NMR and fast-atom-bombardment mass spectrometry have identified it as 2,3-cyclopyrophosphoglycerate, an intramolecularly cyclized pyrophosphate of 2,3-diphosphoglycerate. Chemical degradation to 2,3-diphosphoglycerate and synthesis by dicyclohexylcarbodiimide coupling of 2,3-diphosphoglycerate are consistent with this identification. It is suggested that this compound serves as a primary phosphagen in methanogens.