To understand the role of the puroindolines (PIN-a and PIN-b) in the defense mechanism and stabilization of lipid films in the gas cell of bread dough, we have isolated the proteins and lipids from wheat seed endosperm and studied their interaction at the air/water interface using a Langmuir trough. The nature and shape of the pressure-area compression isotherms of the lipid monolayer in the presence of puroindolines in the subphase depended on the concentration of protein. A distinct phase separation occurred, when the concentration of protein in the subphase increased. The interfacial elasticity of the lipid monolayer in the presence of puroindolines in the subphase was higher than the pure lipid. Injection of protein beneath the preexisting lipid monolayer resulted in the increase of surface pressure due to the penetration of proteins. The extent of penetration depended on the nature of lipid head groups as well as on the initial surface pressure. The penetration of puroindolines to lipid monolayer was observed to be zero after crossing a critical initial surface pressure. The magnitude of the critical initial surface pressure for anionic lipids was significantly higher than the zwitterionic and nonionic lipids. The experimental results showed that both PIN-a and PIN-b had more affinity for anionic polar lipids than the neutral polar lipids and stabilized the lipid monolayer.