The GTPase activity of dynamin is obligatorily coupled, by a mechanism yet unknown, to the internalization of clathrin-coated endocytic vesicles. Dynamin oligomerizes in vitro and in vivo and both its mechanical and enzymatic activities appear to be mediated by this self-assembly. In this study we demonstrate that dynamin is characterized by a tetramer/monomer equilibrium with an equilibrium constant of 1.67 × 1017 M−3. Stopped-flow fluorescence experiments show that the association rate constant for 2′(3′)-O-N-methylanthraniloyl (mant) GTP is 7.0 × 10−5 M−1 s−1 and the dissociation rate constant is 2.1 s−1, whereas the dissociation rate constant for mantdeoxyGDP is 93 s−1. We also demonstrate the cooperativity of dynamin binding and GTPase activation on a microtubule lattice. Our results indicate that dynamin self-association is not a sufficient condition for the expression of maximal GTPase activity, which suggests that dynamin molecules must be in the proper conformation or orientation if they are to form an active oligomer.