Dynamin is a 100 kDa GTPase required for endocytic-coated vesicle formation. Recombinant human neuronal dynamin (dynamin-1) was used for monoclonal antibody (mAb) production. Two mAbs, designated hudy-2 (for human dynamin) and hudy-4, were chosen for further study based on their differential ability to recognize dynamin-1 and its non-neuronal isoform, dynamin-2. Both bind to the proline-rich C-terminal domain (PRD) of dynamin and inhibit the ability of microtubules and grb2 to stimulate GTPase activity. Hudy-4 binds to an epitope within the last 20 amino acids of dynamin-1 and has no effect on its intrinsic GTPase activity. Hudy-2 binds to an epitope within amino acids 822-838 that is common to dynamin-1 and dynamin-2. Hudy-2 stimulates dynamin's intrinsic GTPase activity in a manner proportional to the valency of the immunoglobulin (Ig) G. Crosslinking IgGs with secondary antibodies caused a 2-fold increase in GTPase activity, while F(ab)s were inactive. Importantly, our findings suggest that the stimulation of dynamin GTPase activity by multivalent proteins which bind in vitro to the PRD may not be a valid criterion on its own for assessing the in vivo functional significance of these interactions.