A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle formation from distinct intracellular organelles. There are conflicting data as to a role for dynamin-2 in vesicle budding from the TGN. To resolve this issue, we compared the effects of overexpression of dominant-negative mutants of dynamin-1 (the neuronal isoform) and dynamin-2 (the ubiquitously expressed isoform) on endocytic and biosynthetic membrane trafficking in HeLa cells and polarized MDCK cells. Both dyn1(K44A) and dyn2(K44A) were potent inhibitors of receptor-mediated endocytosis; however neither mutant directly affected other membrane trafficking events, including transport mediated by four distinct classes of vesicles budding from the TGN. Dyn2(K44A) more potently inhibited receptor-mediated endocytosis than dyn1(K44A) in HeLa cells and at the basolateral surface of MDCK cells. In contrast, dyn1(K44A) more potently inhibited endocytosis at the apical surface of MDCK cells. The two dynamin isoforms have redundant functions in endocytic vesicle formation, but can be targeted to and function differentially at subdomains of the plasma membrane.