Abstract Cyclic 2,3-diphosphoglycerate (cDPG), a metabolite exclusively present in methanogenic bacteria, was separated from other phosphate-containing compounds in extracts of Methanobacterium thermoautotrophicum by means of isotachophoresis. The detection limit was 100 pmol per injected sample. Quantification of intracellular concentrations of cDPG with the isotachophoretic assay and with a spectrophoto-metric cDPG assay gave comparable results, but the isotachophoretic assay was faster, less laborious and more sensitive. A possible involvement of cDPG in the energy metabolism was indicated by studies of Mb. thermoautotrophicum in batch and continuous cultures.