Regarding the molecular mechanism of dynamin in receptor-mediated endocytosis, GTPase activity of dynamin has been thought to have a critical role in endocytic vesicle internalization. However, a recent report suggested that GTP-binding to dynamin itself activates the dynamin to recruit molecular machinery necessary for endocytosis. In this study, to investigate the role of GTP binding to dynamin II, we generated two mutant dynamin II constructs: G38V and K44E, G38V, its GTP binding site might be mainly occupied by GTP caused by reduced GTPase activity, and K44E mutant, its GTP binding site might be vacant, caused by its decreased affinity for GTP and GDP. From the analysis of the ratio of GTP vs GDP bound to dynamin, we confirmed these properties. To test the effect of these mutant dynamins on endocytosis, we performed flow cytometry and confocal immunofluorescence analysis and found that these two mutants have inhibitory effect on transferrin-induced endocytosis. Whereas fluorescent transferrin was completely internalized in wild-type (WT), dynamin II expressing cells, no intracellular accumulation of fluorescent transferrin was found in the cells overexpressing K44E and G38V mutant. Interestingly, the amount of GTP bound to K44E was increased when endocytosis was induced than that bound to WT. The present results suggested that the GTPase activity of dynamin II is required for formation of endocytic vesicle and GTP-binding to dynamin II per se is not sufficient for stimulating endocytosis.