Cholesterol is essential for the formation of clathrin-coated endocytic vesicles, as shown by experiments using methyl-β-cyclodextrin (MβCD) to extract cholesterol from the plasma membrane. MβCD treatment significantly inhibited the endocytosis of transferrin and EGF, but had a lesser effect on ricin endocytosis. The inhibition of transferrin endocytosis was reversible, with recovery observed after removal of MβCD and continued incubation in serum-free medium. However, the addition of lovastatin, which inhibits cholesterol synthesis, prevented recovery, suggesting that newly synthesized cholesterol was necessary for restoration. Electron microscopy revealed that MβCD treatment removed cholesterol and disrupted the formation of clathrin-coated pits, leading to the accumulation of shallow coated pits. Despite this, transferrin receptors were still concentrated in coated pits to the same extent as in control cells, indicating that clathrin-independent endocytosis continued. However, cholesterol was essential for the formation of clathrin-coated vesicles. The study also showed that MβCD did not significantly affect plasma membrane permeability or protein synthesis, suggesting that the observed effects were due to cholesterol removal rather than membrane damage. The results indicate that cholesterol is crucial for clathrin-dependent endocytosis, and its removal disrupts the invagination of clathrin-coated pits, thereby impairing vesicle formation. The findings highlight the importance of cholesterol in maintaining the structure and function of clathrin-coated endocytic vesicles.Cholesterol is essential for the formation of clathrin-coated endocytic vesicles, as shown by experiments using methyl-β-cyclodextrin (MβCD) to extract cholesterol from the plasma membrane. MβCD treatment significantly inhibited the endocytosis of transferrin and EGF, but had a lesser effect on ricin endocytosis. The inhibition of transferrin endocytosis was reversible, with recovery observed after removal of MβCD and continued incubation in serum-free medium. However, the addition of lovastatin, which inhibits cholesterol synthesis, prevented recovery, suggesting that newly synthesized cholesterol was necessary for restoration. Electron microscopy revealed that MβCD treatment removed cholesterol and disrupted the formation of clathrin-coated pits, leading to the accumulation of shallow coated pits. Despite this, transferrin receptors were still concentrated in coated pits to the same extent as in control cells, indicating that clathrin-independent endocytosis continued. However, cholesterol was essential for the formation of clathrin-coated vesicles. The study also showed that MβCD did not significantly affect plasma membrane permeability or protein synthesis, suggesting that the observed effects were due to cholesterol removal rather than membrane damage. The results indicate that cholesterol is crucial for clathrin-dependent endocytosis, and its removal disrupts the invagination of clathrin-coated pits, thereby impairing vesicle formation. The findings highlight the importance of cholesterol in maintaining the structure and function of clathrin-coated endocytic vesicles.