Monomolecular films of a 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and any of several nonphospholipids, like diacylglycerols and free fatty acids, inhibit the initial rate of adsorption of an amphipathic protein, colipase, more effectively than the same two-dimensional concentration of phosphatidylcholine alone, even though the non-phospholipids alone have no effect [Sugar, 1. P.; Mizuno, N. K.; Momsen, M. M.; Brockman, H. L. Biophys. J. 2001, 81, 3387-3397]. Inhibition correlates with complex formation between the phosphatidylcholine and non-phospholipid constituents of the monolayer. In the present study, we have examined the formation of complexes of 1,3-dioleoylglycerol with 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine and other species more representative of the phospholipid classes more typically found in the inner leaflet of cellular plasma membranes. These were bovine liver phosphatidylinositol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, -phosphoserine, and -phosphate. Analysis of surface pressure-surface potential-molecular area isotherms showed that all phospholipid species tested were miscible with and formed complexes with 1,3-dioleoylglycerol. Complex compositions were in the range of 0.14-0.25 mole fraction of diacylglycerol. For the phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol mixtures with diacylglycerol at surface pressures near collapse, the initial rate of adsorption of colipase was near zero up to at least the complex composition. With increasing diacylglycerol, the initial adsorption rate increased rapidly to the value observed in the absence of lipid. In contrast, adsorption rates with the phosphatidic acid species increased rapidly beginning at low diacylglycerol mole fractions and were half-maximal at the complex composition. All the data could be analyzed successfully with a statistical model based on the phospholipid exhibiting an excluded area that is independent of overall lipid composition and packing. Values of the excluded areas obtained from the analysis showed an approximate 1 : 1 correspondence with molecular areas of the complexes, assuming no complex for phosphatidic acid. This study shows that phospholipid classes of the type typically exposed to peripheral proteins in the cytoplasm of cells can complex with non-phospholipid second messengers, like diacylglycerols, that are generated in the inner leaflet in response to ligand-receptor interaction on the cell surface. Thus, their complexation with phospholipids has the potential, as for colipase binding, to regulate the translocation. of peripheral proteins to the plasma membrane.