Dynamin is a GTPase protein that is essential for membrane fission during clathrin-mediated endocytosis in eukaryotic cells. Dynasore is a GTPase inhibitor that rapidly and reversibly inhibits dynamin activity, which prevents endocytosis. However, comparison between cells treated with dynasore and RNA interference of genes encoding dynamin, reveals evidence that dynasore reduces labile cholesterol in the plasma membrane, and disrupts lipid raft organization, in a dynamin-independent manner. To explore the role of dynamin it is important to use multiple dynamin inhibitors, alongside the use of dynamin mutants and RNA interference targeting genes encoding dynamin. On the other hand, dynasore provides an interesting tool to explore the regulation of cholesterol in plasma membranes.