Amphiphilic molecules self-assemble at the air-water interface to form insoluble monomolecular layers with the polar headgroup facing water and the hydrophobic region positioned in air. Since GDGTs have two polar head groups we were curious to test how they self-assemble at the air-water interface and therefore understand how flexible these molecules are conformationally. Using a Langmuir-Blodgett trough, we found that GDGT lipids form stable monomolecular layers at the air-water interface as evident from measurements of surface pressure-area (π-A) isotherms obtained by the Wilhelmy plate method (Figure , Figure ). The isotherms exhibited stable liquid expanded behavior lacking any first order transitions. In the case of P2, P1Glc, PG2, and PG1Glc, the surface pressure reached a plateau at significantly high values of 36 mN/m, 30 mN/m, 42 mN/m, and 41 mN/m respectively, which is indicative of monolayer stability.