Bilayer and monolayer phase behavior at the air-water interface of dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylinositol (DPPI), and their binary mixtures with dipalmitoylphosphatidylcholine (DPPC), in the context of their possible roles in lung surfactant function, have been systematically compared. Surface properties, as a function of temperature and composition, were evaluated and analyzed by measuring equilibrium surface pressure-area isotherms (pi -A), equilibrium monolayer collapse pressure (pi (c)), and minimum area per molecule at the monolayer collapse pressure (A(c)) and by direct observation using fluorescence microscopy. Bilayer properties were evaluated by measuring the main bilayer phase transition temperature (T-m) and the bilayer equilibrium spreading pressure (pi (e)) as a function of temperature and composition. Through thermodynamic analysis, it was found that DPPC/DPPG mix ideally in both the monolayer and bilayer states, whereas DPPC/DPPI (further supported with DPPC/Soy PI) are phase-separated with partial miscibility in bilayers and miscible with very significant positive deviations from ideality in monolayers. This behavior is attributed to the distinct differences between PG and PI headgroup properties as reflected in their physical size, state of hydration, and possible conformational flexibility, despite identical net negative charge and identical acyl chain headgroup properties. PC and PG exhibit very similar headgroup properties, which allow the dipalmitoyl chain properties to dominate over headgroup effects in bilayers and monolayers. In contrast, the unique PI headgroup properties dominate over the dipalmitoyl chain effects giving rise to very different surface and bilayer phase behavior.