The bulk of the lipid layer overlying the aqueous portion of the precorneal tear film is composed of polar and nonpolar components. The nonpolar lipids have been the subject of numerous studies; however, the polar lipids have remained relatively uncharacterized. The polar lipids are thought to contain surfactant phospholipids that are critical to the spreading of a lipid film over the aqueous layer, by providing an interface between this layer and the nonpolar lipids. The purpose of the present study is to identify and quantitate the phospholipid complement of meibomian gland secretion which provides the tear film with phospholipids. Meibomian gland secretion was collected from rabbits and phospholipids identified and quantitated by 31P nuclear magnetic resonance spectroscopy. Ten phospholipids were detected from meibomian gland secretion: diphosphatidylglycerol, dihydrosphingomyelin, ethanolamine plasmalogen, phosphatidylethanolamine (PE), phosphatidylserine, sphingomyelin, lysophosphatidylcholine, phosphatidylinositol, alkylacylphosphatidylcholine, and phosphatidylcholine (PC). The two major phospholipids were PC and PE, together comprising nearly 60% of the total phospholipid profile. The nature and relative concentrations of the meibomian gland secretion phospholipids are congruous with a surfactant role at the aqueous-lipid interface and, considering the physical chemistry of the tear film, suggest that the phospholipids should be organized in a very flat or planar configuration.