The ability of mouse peritoneal macrophages to hydrolyze and excrete cytoplasmic cholesteryl ester droplets was studied. The macrophages were loaded with cholesteryl esters by incubation with acetylated low density lipoprotein (acetyl-LDL), which is internalized by adsorptive endocytosis. The cholesteryl esters of acetyl-LDL are hydrolyzed within lysosomes and the liberated cholesterol is re-esterified in the cytoplasm where it accumulates as cytoplasmic cholesteryl ester droplets. Hydrolysis and excretion of these stored cholesteryl esters were quantified by gas-liquid chromatographic measurement of the content of free and esterified cholesterol in cells and in medium. After removal of acetyl-LDL from the culture medium, the cytoplasmic cholesteryl esters were rapidly hydrolyzed and large amounts of free cholesterol were excreted from the cells. Hydrolysis and excretion required a cholesterol acceptor in the culture medium. The following agents were shown to be effective as cholesterol acceptors: high density lipoprotein (HDL), whole serum, the density > 1.215 g/ml fraction of whole serum, intact erythrocytes, casein, and thyroglobulin. The following agents did not promote the hydrolysis and excretion of cholesteryl esters under these experimental conditions: LDL, serum albumin, serum gamma-globulins, and phosphatidylcholine/sphingomyelin liposomes. The results indicate that net hydrolysis of cytoplasmic cholesteryl esters in macrophages is coupled to the process of cholesterol excretion and that net hydrolysis does not occur unless an effective cholesterol acceptor is present in the culture medium.-Ho, Y. K., M. S. Brown, and J. L. Goldstein. Hydrolysis and excretion of cytoplasmic cholesteryl esters by macrophages: stimulation by high density lipoprotein and other agents.