The GTPase dynamin is required for endocytic vesicle formation. Dynamin has also been implicated in regulating the actin cytoskeleton, but the mechanism by which it does so is unclear [1-4]. Through interactions via its proline-rich domain (PRD), dynamin binds several proteins, including cortactin , profilin , syndapin , and murine Abp1 , that regulate the actin cytoskeleton. We investigated the interaction of dynamin2 and cortactin in regulating actin assembly in vivo and in vitro. When expressed in cultured cells, a dynamin2 mutant with decreased affinity for GTP decreased actin dynamics within the cortical actin network. Expressed mutants of cortactin that have decreased binding of Arp2/3 complex or dynamin2 also decreased actin dynamics. Dynamin2 influenced actin nucleation by purified Arp2/3 complex and cortactin in vitro in a biphasic manner. Low concentrations of dynamin2 enhanced actin nucleation by Arp2/3 complex and cortactin, and high concentrations were inhibitory. Dynamin2 promoted the association of actin filaments nucleated by Arp2/3 complex and cortactin with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing lipid vesicles. GTP hydrolysis altered the organization of the filaments and the lipid vesicles. We conclude that dynamin2, through an interaction with cortactin, regulates actin assembly and actin filament organization at membranes.