The GTPase dynamin plays an essential role in clathrin-mediated endocytosis [1] [2] [3]. Substantial evidence suggests that dynamin oligomerisation around the necks of endocytosing vesicles and subsequent dynamin-catalysed GTP hydrolysis is responsible for membrane fission [4] [5]. The pleckstrin homology (PH) domain of dynamin has previously been shown to interact with phosphoinositides, but it has not been determined whether this interaction is essential for dynamin's function in endocytosis [6] [7] [8] [9]. In this study, we address the in vivo function of the PH domain of dynamin by assaying the effects of deletions and point mutations in this region on transferrin uptake in COS-7 fibroblasts. Overexpression of a dynamin construct lacking its entire PH domain potently blocked transferrin uptake, as did overexpression of a dynamin construct containing a mutation in the first variable loop of the PH domain. Structural modelling of this latter mutant suggested that the lysine residue at position 535 (Lys535) may be critical in the coordination of phosphoinositides, and indeed, the purified mutant no longer interacted with lipid nanotubes. Interestingly, the inhibitory phenotype of cells expressing this dynamin mutant was partially relieved by a second mutation in the carboxy-terminal proline-rich domain (PRD), one that prevents dynamin from binding to the Src homology 3 (SH3) domain of amphiphysin. These data demonstrate that dynamin's interaction with phosphoinositides through its PH domain is essential for endocytosis. These findings also support our hypothesis that PRD-SH3 domain interactions are important in the recruitment of dynamin to sites of endocytosis.