Pulmonary surfactant is the mixture of lipids and proteins that lowers surface tension in the lungs. The material forms a thin film on the aqueous layer that lines the alveolar air-sacks. When compressed by the shrinking alveolar surface area during exhalation, the surfactant film achieves surface tensions < 5 mN/m. The molecular arrangement of the film that sustains these low tensions remains obscure. Vesicles of pulmonary surfactant adsorb as collective packets, delivering their complete set of constituents that initially form monolayers at the interface. At physiological temperatures, such monomolecular films readily collapse from the surface. Additional vesicles added below these initial monolayers substantially increase their stability at low surface tensions. The studies here focus on the structural changes that produce that improved stability. Two divergent models attempt to explain how the additional material enhances the resistance to collapse: enrichment of dipalmitoyl phosphatidylcholine (DPPC) in the interfacial monolayer to form a crystalline film; and formation of a multilayered structure. Surface X-ray scattering determined how calf lung surfactant extract added below monolayers of that material affects the structure of the film. The ordered structure within the initial monolayer differed from the DPPC packing motif, but fit with the hexagonal lattice of a binary mixture with DPPC and cholesterol at a ratio of 4:1 (mol%). Vesicles added to the subphase had no effect on the local structure of the film, which remained monomolecular in thickness with an unchanged hexagonal lattice. The vesicles, however, produced a concentration-dependent increase in the size of individual ordered regions, and in their total area. Our results suggest that the functional film contains cholesterol:DPPC rather than DPPC alone, and that vesicles in the subphase provide a reservoir of additional DPPC that allows the ordered phase to grow.