Dynamin, a 100-kD GTPase, is essential for endocytosis in mammalian cells. To investigate its role, researchers generated stable HeLa cell lines expressing wild-type or mutant dynamin driven by a tetracycline-inducible promoter. Overexpression of wild-type dynamin had no effect, but mutant dynamin blocked coated pit constriction and coated vesicle budding, inhibiting endocytosis via transferrin and EGF receptors. Coated pit assembly and receptor recruitment were unaffected. Other vesicular transport pathways, such as transferrin receptor recycling and biosynthesis, were also normal. EM immunolocalization showed that dynamin was specifically associated with clathrin-coated pits on the plasma membrane. The mutant dynamin accumulated long tubules connected to the plasma membrane. These findings indicate that dynamin is specifically required for endocytic coated vesicle formation, and its GTP binding and hydrolysis activities are essential for this process.Dynamin, a 100-kD GTPase, is essential for endocytosis in mammalian cells. To investigate its role, researchers generated stable HeLa cell lines expressing wild-type or mutant dynamin driven by a tetracycline-inducible promoter. Overexpression of wild-type dynamin had no effect, but mutant dynamin blocked coated pit constriction and coated vesicle budding, inhibiting endocytosis via transferrin and EGF receptors. Coated pit assembly and receptor recruitment were unaffected. Other vesicular transport pathways, such as transferrin receptor recycling and biosynthesis, were also normal. EM immunolocalization showed that dynamin was specifically associated with clathrin-coated pits on the plasma membrane. The mutant dynamin accumulated long tubules connected to the plasma membrane. These findings indicate that dynamin is specifically required for endocytic coated vesicle formation, and its GTP binding and hydrolysis activities are essential for this process.