Triacylglycerols are the major substrates for lipolytic enzymes that act at the surface of emulsion-like particles such as triglyceride-rich lipoproteins, chylomicrons, and intracellular lipid droplets. This study examines the effect of cholesterol on the solubility of a triacylglycerol, triolein, in phospholipid surfaces. Solubilities of [carbonyl-13C]triolein in phospholipid bilayer vesicles containing between 0 and 50 mol % free cholesterol, prepared by cosonication, were measured by 13C NMR. The carbonyl resonances from bilayer-incorporated triglyceride were shifted downfield in the 13C NMR spectra from those corresponding to excess, nonincorporated material. This enabled solubilities to be determined directly from carbonyl peak intensities at most cholesterol concentrations. The bilayer solubility of triolein was inversely proportional to the cholesterol/phospholipid mole ratio. In pure phospholipid vesicles the triolein solubility was 2.2 mol %. The triglyceride incorporation decreased to 1.1 mol % at a cholesterol/phospholipid mole ratio of 0.5, and at a mole ratio of 1.0 for the bilayer lipids, the triolein solubility was reduced to just 0.15 mol %. The effects of free cholesterol were more pronounced and progressive than observed previously on the bilayer solubility of cholesteryl oleate (Spooner, P. J. R., Hamilton, J. A., Gantz, D. L., & Small, D. M. (1986) Biochim. Biophys. Acta 860, 345-353]. As with cholesteryl oleate, we suggest that cholesterol also displaces solubilized triglyceride to deeper regions of the bilayer.