We have investigated the kinetic properties of acid cholesteryl esterase (EC 3.1.1.13) in preparations of rabbit aortic cells, with the aim of establishing conditions suitable for the quantitative assay of the enzyme in freshly prepared homogenates and subcellular fractions, whether derived from normal cells or from atheromatous cells heavily laden with cholesterol and cholesteryl esters. As substrate we used cholesteryl ( l-'4C)oleate incorporated at a 1 : 100 molar ratio into egg-lecithin liposomes as described by Brecher and co-workers (J. Lipid Res. 1976. 17: 239), and measured the radioactivity remaining in the alkaline buffer phase after organic solvent extraction of unhy- drolyzed substrate. When the liposome substrate was used as such, more than 80% of the enzyme activity was latent in fresh homogenates. It could be released quantitatively without inactivation by addition of digitonin, but this created a requirement for taurocholate, presumably because digi- tonin disrupts liposomes. The following conditions gave satisfactory linearity with both time of incubation and en- zyme concentration, with both normal and atheromatous cell preparations, and were adopted for the assay: 12.7 pM cholesteryl oleate dispersed in 1.27 mM egg lecithin; 50 mM acetate buffer, pH 3.9; 2.0 mM Na taurocholate; and 0.005%' digitonin. The enzyme has a pH optimum of 3.9, and, under our conditions, has an apparent Km of about 1.5 pM. It is markedly inhibited by salt solutions and is sensitive to freezing, especially at high ionic strength. Subcellular rractionation by sucrose density gradient cen- trifugation indicates that a considerable part of the enzyme is localized in lysosomes, both in normal and in either moderately or heavily lipid-laden atheromatous aortic cells. The proportion of activity associated with the soluble frac- tion is however higher for acid cholesteryl esterase than for two acid glycosidases, in all three cell preparations. When assayed under optimal conditions, lipid-laden atheromatous cells displayed up to 3.5 times the acid cholesteryl esterase activity of normal aortic cells. This finding does not support the hypothesis that lipid overloading of lysosomes in atheromatous arterial cells occurs as a consequence of a relative deficiency of acid cholesteryl esterase.-Haley, N. J., S. Fowler, and C. de Duve. Lysosomal acid cholesteryl esterase activity in normal and lipid-laden aortic cells. J. Lipid Rr.c.. 1980. 21: 961-969.