Our studies reveal the presence of a variety of archaeal and bacterial biomarkers at the Lost City hydrothermal field. In the porous outer parts of the carbonate chimneys, variations in hydrothermal fluid flow allow for more or less extensive seawater infiltration, creating an interface where specific microbial biofilm communities can develop. Microbiological data together with the lipid distributions and their compound-specific carbon isotope compositions indicate that this habitat primarily supports CH4-oxidizing bacteria and the CH4-cycling archaeon LCMS.This study reveals the extraordinary heterogeneity in the C25 and C30 polyisoprenoid hydrocarbon (PMIs and squalenoids) distributions and in their d13C composition, showing values ranging from -11.4& to +24.6&. PMIs and squalenoids at Lost City were found to derive from the LCMS archaea. Variations in d13C values in archaeal biomarkers most probably deriving from single species could result from unusual isotopic fractionation in this particularly stressful environment. However, the diversity in PMI and squalenoid distributions and d13C values is rather proposed to reflect the ability of the LCMS archaea to adapt to conditions where carbon is limited. The specific biofilm mode of growth of the archaeal LCMS likely contributes to the heterogeneity observed in the lipid distributions. The integration of microbiological studies and archaeal biomarker data also indicates that the extent of seawater mixing with the hydrothermal fluids may be recorded in the relative abundance of the unusual H-shaped GDGT, possibly produced in the subseafloor by Thermoccocales.The LCMS archaea may be capable of CH4 production and consumption as a mechanism to adapt to the different extreme biochemical niches encountered at Lost City. We argue that the structural diversity in the PMIs/squalenoids detected in our study probably reflects this versatility. A deeper knowledge of the physiological role of these specific lipid biomarkers may help to unravel the question as to why so many different PMIs and squalenoids are produced and in response to which factors.