NECAPs (adaptin ear-binding clathrin-associated protein) are a new family of clathrin accessory proteins identified through a proteomic analysis of clathrin-coated vesicles (CCVs) from the brain. One member of this family, NECAP 1, is found primarily in tissues from the central nervous system and has been shown to be complexed tightly with a substantial portion of adaptor protein-2 (AP-2) in brain extracts. However, the function and intracellular location of this protein is unknown. In this study, we find that endogenous and epitope-tagged NECAP 1 co-localizes well with clathrin and AP-2 in punctate structures, many of which also contain the presynaptic markers synaptophysin, synaptotagmin or synaptic vesicle protein 2 (SV2). NECAP 1 was also detected by western blot in synaptic vesicle preparations. Overexpression of a truncation mutant of NECAP 1 (BC-NECAP 1) in neurons inhibited transferrin endocytosis but not epidermal growth factor (EGF) endocytosis, and this inhibition was dependent on an AP-2-binding WVQF motif. Moreover, overexpression of BC-NECAP 1 results in inhibition of synaptotagmin endocytosis both in unstimulated neurons and in neurons stimulated with potassium chloride. This inhibition was abrogated by truncation of the WVQF domain. We conclude from these observations that NECAP 1 plays a role in clathrin-mediated neuronal endocytosis, including a role in presynaptic endocytosis.