The size of the polar head group plays a key role in lipid biophysics, influencing membrane organization, phase behavior, including transition (melting) temperature, as well as lipid–lipid interactions, such as the umbrella effect. In general, small head groups are associated with high transition temperatures and tighter head group packing compared to lipids with large head groups. The head groups of phosphatidylglucoside (PtdGlc) and phosphatidylinositol (PtdIns) are configurational isomers with both featuring a six-membered ring and a similar number of hydroxy functions. Interestingly, their chemical stability and biophysical properties differ significantly, despite their high structural similarity, comparable chemical size and weight, and presumably analogous head group orientation relative to the plasma membrane normal. PtdIns is known to exhibit a low transition temperature, limited lipid–lipid head group interaction, a high degree of head group hydration and an intramolecular hydrogen-bond even under fully hydrated conditions. PtdGlc on the other hand exhibits a ~20 degree higher transition temperature, extensive lipid–lipid head group interactions, a low degree of head group hydration and no intramolecular hydrogen-bond. Consequently, head group size in terms of lipid biophysics, refers not only to the immediate chemical size, but also includes the primary hydration shell.