Lipid molecules self-assemble into supramolecular structures called mesophases (e.g. lamellar, hexagonal, cubic) depending on their molecular geometry and external factors. Functional roles of lipids are dictated by their propensity to form specific mesophases. For example, the tendency of phosphoethanolamine lipids to form inverted non-lamellar phases has been associated with their role in facilitating fusion of biological membranes via induction of negative curvature. SAXS on bulk lipid dispersions produces characteristic diffraction patterns from which specific mesophases can be ascertained (Figure ). We sought to establish a link between the mesophase behavior of the lipids across a wide temperature range and their probable functional roles in archaeal cell membranes. Differential scanning calorimetry (DSC) studies on bulk GDGT lipid mixtures from extracts typically revealed that the lipids exist in fluid phases at all temperatures and do not exhibit any endothermic phase transitions. However, it may be challenging to detect transitions like lamellar to hexagonal which are typically characterized by low enthalpy changes. Moreover, each DSC measurement requires a significant amount of material that is not always practical to recover. Therefore, we characterized the mesophases formed by aqueous GDGT dispersions using synchrotron SAXS over 10-90 °C under the influence of (i) varying salt concentration; (ii) presence of Mg 2+ ; and (iii) acidic pH. The results are summarized in Table . In a low ionic strength buffer such as 50 mM Tris (pH 7.5), P2, PG2, and PG1Glc exhibited no Bragg peaks (Figure , Figures ) over the entire temperature range while P1Glc exhibited a weak hexagonal phase at 30 °C which gradually increased in intensity with temperature (Figure ). These results suggest that the lipids mostly formed uncorrelated bilayers in Tris buffer due to repulsive forces between the charged headgroups. Repulsive forces may also arise from mutual steric hindrance between undulating membranes stacked in a liquid crystalline multilayer. Addition of salts is expected to screen the electrostatic repulsion and generate more ordered structures. Therefore, we investigated the effect of 0.1 M, 0.5 M, and 1 M NaCl added to 50 mM Tris buffer (pH 7.5). Among all lipids, P2 exhibited the most complex behavior at varying salt concentrations (Figure ). In presence of 0.1 M NaCl, somewhat broad and low intensity HII peaks were observed at 10-20 °C which became sharper and more intense over 30-70 °C. Over 80-90 °C, an Pn3m cubic phase coexisted with the HII phase (Figure ). In 0.5 M NaCl, strong lamellar peaks were seen at 10 °C which fully transitioned to HII peaks at 60 °C and above via an la3d cubic phase at 50 °C. In 1.0 M NaCl, strong lamellar peaks existed over 10-30 °C which coexisted with an HII phase at 40-60 °C and an HII phase only was observed at higher temperatures. P1Glc was difficult to disperse even in the presence of 0.1 M NaCl and a hexagonal phase was detectable at 50 °C and above (Figure ). P1Glc film could not be hydrated at higher salt concentrations. PG2 exhibited disordered phases at all salt concentrations except a small degree of lamellar ordering over 10-30 °C in the presence of 1 M NaCl (Figure ). These observations are in good agreement with microscopy studies where we see facile formation of PG2 vesicles in buffer having a similar range of ionic strengths. PG1Glc displayed disordered phases in 0.1 M NaCl, but a hexagonal phase started to appear above 30 °C in the presence of 0.5 M NaCl (Figure ). In the presence of 1 M NaCl, a small hexagonal phase peak was detectable even at 10 °C which increased in intensity with temperature (Figure ).