The study investigates the role of cholesterol in endocytosis using methyl-β-cyclodextrin (MβCD) to selectively extract cholesterol from the plasma membrane. MβCD treatment strongly inhibits endocytosis of transferrin and EGF but has a less significant effect on ricin endocytosis. The inhibition of transferrin endocytosis is reversible upon removal of MβCD, suggesting that it is due to cholesterol depletion. Electron microscopy reveals that MβCD treatment disrupts the formation of clathrin-coated pits, leading to the accumulation of shallow coated pits. Quantitative immunogold labeling shows that transferrin receptors are concentrated in these shallow coated pits, indicating that cholesterol is essential for the invagination of clathrin-coated pits. The results suggest that cholesterol is crucial for clathrin-dependent endocytosis, while clathrin-independent endocytosis remains functional despite cholesterol depletion.The study investigates the role of cholesterol in endocytosis using methyl-β-cyclodextrin (MβCD) to selectively extract cholesterol from the plasma membrane. MβCD treatment strongly inhibits endocytosis of transferrin and EGF but has a less significant effect on ricin endocytosis. The inhibition of transferrin endocytosis is reversible upon removal of MβCD, suggesting that it is due to cholesterol depletion. Electron microscopy reveals that MβCD treatment disrupts the formation of clathrin-coated pits, leading to the accumulation of shallow coated pits. Quantitative immunogold labeling shows that transferrin receptors are concentrated in these shallow coated pits, indicating that cholesterol is essential for the invagination of clathrin-coated pits. The results suggest that cholesterol is crucial for clathrin-dependent endocytosis, while clathrin-independent endocytosis remains functional despite cholesterol depletion.