The content of the meibomian gland lipid exprimate is known, but little is known about the phospholipids that comprise the glandular cells. The purpose of the present study is to identify and quantitate the phospholipid complement of the meibomian gland cells that produce the lipid secretion of meibomian oil and which is vital to tear film stability.Eyelids (n = 50) were excised from rabbits, and after surgical removal of surrounding tissues, the tarsal plates with and without expressing meibomian oil were extracted and phospholipids of the plates quantified by31P nuclear magnetic resonance (NMR).Seventeen phospholipids were quantified from tarsal plates expressed of oil and tarsal plates containing meibomian oil: alkylacylphosphatidylcholine (AAPC), dihydrosphingomyelin (DHSM), dimethylphosphatidylethanolamine, diphosphatidyl-glycerol (cardiolipin), ethanolamine plasmalogen (EPLAS), lyso-ethanolamine plasmalogen, lysophosphatidylcholine, lysophos-phatidylethanolamine, lysophosphatidylserine, phosphatidic acid, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, sphingomyelin (SM), sphingosylphosphorylcholine. The six zwit-terionic and neutral phospholipids, DHSM, EPLAS, PE, SM, AAPC, and PC together comprise 79.5% of the total meibomian gland phospholipid profile (in meibomian oil this value is 84.2%). The zwitterionic and neutral phospholipids dominate meibomian gland phospholipid profiles. Since the meibomian gland cells undergo holocrine secretion and form the meibomian gland secretion, such a composition is consistent with the hypothesis that a chemically stable lamellar surfactant layer phospholipids bind non-polar meibomian oil to the aqueous layer of the tear film.