Archaeal membranes show a great structural variety, recorded in lipid biomarkers, which is thought to reflect adaptation to physiological and evolutionary environmental conditions, such as pH and temperature. Distribution of archaeal lipids might also depend on nutrient availability, source of energy, or level of oxygen. Lipid biomarkers in CH4-metabolizing archaea mainly include C25 and C30 isoprenoid hydrocarbons as well as glycerol di- and tetraethers with C20 or C40 isoprenoid and hydroxylated isoprenoid moieties. Here, we focus on archaeal biomarkers from young carbonate–brucite deposits that were collected in 2003 and 2005 at various actively venting sites across the LCHF (Table 1, Fig. 1). The 2005 samples were collected from the surface of the deposits and hence are expected to record interface processes between hydrothermal fluids and seawater in the outer part of the chimneys. Our goal is to use molecular biomarker distributions and compoundspecific carbon isotopes in association with genetic data to better understand archaeal activity at the surface of the Lost City carbonate chimneys, whereas the previous study on the molecular biomarker distributions at the LCHF focused on whole pieces of carbonate towers that are expected to reflect mainly the microbial activity in the interior part of the carbonate chimneys.