Archaea are an enigmatic group of prokaryotic organisms. Originally thought to be extremophiles, archaea have been found in every realm of the biosphere including the human microbiome. 1 Archaea constitute ~1.3% of the total biomass on the Earth, 2 yet they have remained quite underexplored. While archaea resemble bacteria in certain superficial ways, fundamental differences in their biochemistry led to their classification as a separate domain of life. For instance, archaeal lipids differ significantly from the lipids found in bacteria (and eukaryotes) in terms of structural features, such as, absolute stereochemistry of the glycerol moiety, the nature of the hydrocarbon chains and their linkages to the glycerol moiety. These distinctions between the lipids of Archaea and Bacteria (and Eukarya) form the basis of a dichotomy known as the "Lipid Divide" (Figure ). In archaeal lipids, the hydrocarbon chains have multiple methyl branches and are of isoprenoid origin -typically lacking any unsaturation. Their hydrocarbon chains are connected to the sn-glycerol-1-phosphate backbone via ether linkages. In bacterial lipids, the hydrocarbon chains are fatty acidbased, contain various degrees of unsaturation, and the hydrocarbon chains are connected to the sn-glycerol-1-phosphate backbone through ester linkages. Lack of unsaturation and the presence of ether linkages make the archaeal lipids extremely stable to aerial oxidation and hydrolysis respectively. Due to this exceptional chemical stability, archaeal lipids serve as excellent biomarkers for biogeochemical and climate studies. Remarkably, a major class of archaeal lipids has a bipolar structure -which is analogous to two monopolar lipids covalently joined at the ends of their hydrocarbon tails. The bipolar nature of such archaeal lipids gives rise to the question of how these molecules are organized within a membrane? Three types of limiting molecular arrangements can be imagined (Figure ): (1) stretched -the lipid molecules are fully stretched (i.e., O-shaped) and span the entire width of the membrane to form a monolayer; (2) looped -the lipid molecules are bent (i.e., U-shaped) at the hydrocarbon region so that the two headgroups face the same side of the membrane, so the membrane more closely resembles a bilayer; and (3) mixed -where both O-and U-shaped lipids coexist. There is considerable evidence that suggests that at least a majority of the lipids occur in a stretched conformation. For instance, in contrast to bilayer membranes, freeze-fracture electron microscopy studies on archaeal tetraether lipid vesicles have revealed the absence of a preferential fracture plane, which can be explained by considering that the lipids adopt a stretched conformation. Although there is a report mentioning exclusive formation of U-shaped bilayers on a solid substrate via transfer of Langmuir films, evidence of such structures in biological membranes or model vesicles is lacking. Therefore, it is generally assumed that the archaeal bipolar lipids adopt a membrane-spanning conformation, although the possibility of a small percentage of U-shaped lipids coexisting with O-shaped lipids has been speculated. Figure . A. Schematic diagram illustrating the concept of the "Lipid Divide" between bacterial/eukaryotic and archaeal lipids. The carbons on glycerol moieties are marked (1-3) according to stereochemical numbering (sn) convention. (R) and (S) denote the absolute stereochemistries of the glycerol moieties. The blue spheres denote phosphate-containing polar headgroups. For simplicity, the stereochemistries of the branched chains are not shown. B. Three possibilities of shapes adopted by archaeal bipolar lipids in a membrane.