The incorporation of cholesteryl esters (CE) with saturated acyl chains into the lamellar structure of phospholipids was studied by 13C nuclear magnetic resonance (NMR) spectroscopy. The CE (octanoate, palmitate, and stearate) were 13C-enriched in the carboxyl carbon to enhance the signals of the small amounts of bilayer-incorporated CE. Magic-angle spinning (MAS) NMR was used to detect signals in unsonicated multilamellar phosphatidylcholine (PC), and solution NMR was used to detect signals in PC small unilamellar vesicles (SUV). All CE showed a carbonyl peak reflecting localization of the carbonyl at the aqueous interface (Hamilton & Small, 1982). The maximal incorporation decreased with chain length, from 5 mol % for octanoate to 1.4 mol % for stearate in multilayers; the stearate ester had a solubility slightly lower than that of cholesteryl oleate (2 mol %). The maximal incorporation of a specific CE was 1.2-2.0 times higher in SUV than in multilayers. Cholesteryl oleate did not prevent solubilization of cholesteryl stearate in the PC interface. CE in excess of the solubility maximum in multilayer samples was shown to be crystalline at 35 degrees C by MAS NMR; thus, in the bilayer the CE gained considerable mobility compared to its mobility in its pure bulk phase at body temperature. Furthermore, CE with two saturated chains were not distinguishable from esters with an unsaturated chain with respect to mobility and position in the interface. These fundamental interfacial properties assure utilization of CE with common dietary fatty acyl chains.