Stimulation of intracellular cholesterol esterification, which is catalyzed by the enzyme acyl-coenzyme A: cholesterol O-acyltransferase (ACAT), by atherogenic lipoproteins in macrophages is a key step in the ability of these cells to store lipoprotein-cholesterol and in the eventual development of atheroma foam cells. Herein, we provide evidence that the actin cytoskeleton plays an important role in the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages. When the actin cytoskeleton of cultured mouse peritoneal macrophages was disrupted by treatment with cytochalasin D or Clostridial C2 toxin, the ability of beta very low density lipoprotein (beta-VLDL) to stimulate cholesterol esterification was decreased 3-6-fold, even under conditions in which beta-VLDL protein degradation, cholesteryl ester hydrolysis, or net cholesterol delivery to the cells was matched. Esterification of cellular phospholipids and triglycerides was not affected by this treatment. Cytochalasin D treatment of macrophages also inhibited the ability of acetyl-low density lipoprotein, another foam cell-forming lipoprotein, to stimulate cholesterol esterification, but stimulation of cholesterol esterification by 25-hydroxycholesterol was not inhibited by cytochalasin D. Cytochalasin D was found to inhibit neither the exit of beta-VLDL-derived cholesterol from lysosomes nor the ability of beta-VLDL to down-regulate endogenous cholesterol synthesis. From these data we conclude that an intact actin cytoskeleton is necessary for efficient stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages. Although the exact function of actin in the cholesterol esterification pathway remains to be determined, our data rule out a general role for actin in intracellular cholesterol trafficking or maintenance of ACAT enzyme activity. Rather, we speculate that actin filaments play a role in specific cellular entry processes of atherogenic lipoproteins and/or in establishing transport or contact between the plasma membrane cholesterol substrate pool and the ACAT enzyme in macrophages.