The function of the dopamine transporter (DAT) to terminate dopamine neurotransmission is regulated by endocytic trafficking of DAT. To elucidate the mechanisms of DAT endocytosis, we generated a fully functional mutant of the human DAT in which a hemagglutinin epitope (HA) was incorporated into the second extracellular loop. The endocytosis assay, based on the uptake of an HA antibody, was designed to study constitutive- and protein kinase C (PKC)-dependent internalization of HA-DAT expressed in non-neuronal cells and rat dopaminergic neurons. Large-scale RNA interference analysis of PKC-dependent endocytosis of HA-DAT revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4–2 (neural precursor cell expressed, developmentally downregulated 4–2), as well as the involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15 (epidermal growth factor pathway substrate clone 15), and Eps15R (Eps15-related protein). Depletion of Nedd4–2 resulted in a dramatic reduction of PKC-dependent ubiquitination of DAT. Endogenous Nedd4–2, epsin, and Eps15 were coimmunoprecipitated with heterologously expressed human HA-DAT and endogenous DAT isolated from rat striatum. A new mechanistic model of DAT endocytosis is proposed whereby the PKC-induced ubiquitination of DAT mediated by Nedd4–2 leads to interaction of DAT with adaptor proteins in coated pits and acceleration of DAT endocytosis.