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Summary Butanetriol dialkyl glycerol tetraethers (BDGTs) are a novel class of archaeal membrane lipids possibly related to methanogenesis. Their structure deviates from the other archaeal lipid structures, such as the glycerol dialkyl glycerol tetraethers (GDGTs), as they possess a four-carbon backbone instead of the universal glycerol moiety. Here, we combined compound-specific stable isotope probing and high-resolution mass spectrometry to investigate the biosynthetic pathway of BDGTs. We demonstrated that BDGTs result from a post-synthesis methylation of GDGTs. We further observed that biosynthesis of BDGTs was stimulated by energy and/or nutrient depletion both in vitro and in the environment. We hence posit that production of BDGTs act as an adaptation strategy to survive in energy or nutrient deprived environments such as in the deep sub-seafloor.