Human meibomian gland secretions (also known as meibum) were analyzed for the presence of cholesteryl esters (Chl-E) using HPLC in combination with atmospheric pressure chemical ionization mass spectrometry. A special procedure based on detection of the in-source generated ion m/z 369 was developed to monitor all Chl-E simultaneously. The structures of the detected compounds were studied using in-source and postsource fragmentation of the precursor (M+H)(+) ions. In concordance with previous studies, Chl-E were found in all of the tested samples and comprised similar to 31% of the entire lipid pool (w/w, dry weight). There were at least 20 different saturated and unsaturated Chl-E species observed, whose fatty acid residues ranged from C18 to C34. Monounsaturated fatty acids were the most visible components of the Chl-E pool. The eleven most prominent compounds were: C20:0-, C22:1-, C22:0-, C24:1-, C24:0, C25:0-, C26:1-, C26:0-, C28:1-, C28:0-, and C30:1-Chl-E. Other Chl-containing compounds were detected but not identified at the time. Therefore, Chl-E are a depot for very long chain saturated and monounsaturated fatty acids in human meibum.-Butovich, I. A. Cholesteryl esters as a depot for very long chain fatty acids in human meibum. J. Lipid Res. 2009. 50: 501-513.