PURPOSE. The purpose of this investigation was to determine the major molecular components of the lipids in normal human meibomian gland secretions (meibum). METHODS. The meibum samples were studied by direct infusion electrospray ionization (ESI), quadrupole time-of-flight mass spectrometry, and tandem mass spectrometry (MS/MS) analysis, in both positive and negative detection modes. RESULTS. Hundreds of peaks were detected, among which the molecular compositions and subclasses of approximately 160 major peaks were confidently identified. The compositions and subclasses of these peaks were determined from collision-induced dissociation fragmentation patterns, highresolution and high-mass-accuracy spectra, and references of literature reports. The major peaks detected in positive mode were those of nonpolar lipids, including wax esters, cholesteryl esters, triacylglycerols, and diesters, whereas in negative mode, the major peaks detected were those of polar lipids, including free fatty acids and (O-acyl)--hydroxy fatty acids. CONCLUSIONS. The analysis of intact lipids in meibum with direct infusion ESI-MS/MS analysis has the advantages of minimal sample preparation (no chromatography or pre-separation needed), mild experimental conditions, high throughput, and high sensitivity. © Association for Research in Vision and Ophthalmology.