Abstract The anaerobic oxidation of methane (AOM) is a microbial process present in marine and freshwater environments. AOM is important for reducing the emission of the second most important greenhouse gas methane. In marine environments anaerobic methanotrophic archaea (ANME) are involved in sulfate-reducing AOM. In contrast, Ca . Methanoperedens of the ANME-2d cluster carries out nitrate AOM in freshwater ecosystems. Despite the importance of those organisms for AOM in non-marine environments not much is known about their lipid composition or carbon sources. To close this gap, we analyzed the lipid composition of ANME-2d archaea and found that they mainly synthesize archaeol and hydroxyarchaeol as well as different (hydroxy-) glycerol dialkyl glycerol tetraethers, albeit in much lower amounts. Abundant lipid headgroups were dihexose, monomethyl-phosphatidyl ethanolamine and phosphatidyl hexose. Moreover, a monopentose was detected as a lipid headgroup which is rare among microorganisms. Batch incubations with 13 C labelled bicarbonate and methane showed that methane is the main carbon source of ANME-2d archaea varying from ANME-1 archaea which primarily assimilate dissolved inorganic carbon (DIC). ANME-2d archaea also assimilate DIC, but to a lower extent than methane. The lipid characterization and analysis of the carbon source of Ca. Methanoperedens facilitates distinction between ANME-2d and other ANMEs.