Abstract A highly sensitive method for the quantification of methanogens in anaerobic digestor sludges was developed, based on an analysis of ether-linked glycerolipids. Core lipids were prepared from total lipids by HF treatment and mild methanolysis, and these core lipids were quantified as the corresponding 9-anthroyl derivatives by high-performance liquid chromatography with fluorescence detection. The amounts, in terms of cell carbon content, of Methanosaeta and Methanosarcina were proportional to the amounts of α-hydroxyarchaeol and β-hydroxyarchaeol, respectively. Moreover, the total amount of core lipids was well correlated with the cell mass of aceticlastic and H2/CO2-consuming methanogens. The limit of detection for Methanosaeta concilii was 17 ng of cell carbon when the signal/noise ratio was 3. This method allowed us to quantitate aceticlastic methanogens with high accuracy and to make a rough estimate of total methanogenic cells without any interference by the multifarious impurities that are present in anaerobic sludges. These results suggest that the present method will be a useful tool for investigations of methanogenic ecosystems.