Dopamine levels in the brain are controlled by the plasma membrane dopamine transporter (DAT). The amount of DAT at the cell surface is determined by the relative rates of its internalization and recycling. Activation of protein kinase C (PKC) leads to acceleration of DAT endocytosis. We have recently demonstrated that PKC activation also results in ubiquitylation of DAT. To directly address the role of DAT ubiquitylation, lysine residues in DAT were mutated. Mutations of each lysine individually did not affect ubiquitylation and endocytosis of DAT. By contrast, ubiquitylation of mutants carrying multiple lysine substitutions was reduced in cells treated with phorbol ester to the levels detected in nonstimulated cells. Altogether, mutagenesis data suggested that Lys19, Lys27, and Lys35 clustered in the DAT amino-terminus are the major ubiquitin-conjugation sites. The data are consistent with the model whereby at any given time only one of the lysines in DAT is conjugated with a short ubiquitin chain. Importantly, cell surface biotinylation, immunofluorescence and down-regulation experiments revealed that PKC-dependent internalization of multilysine mutants was essentially abolished. These data provide the first evidence that the ubiquitin moieties conjugated to DAT may serve as a molecular interface of the transporter interaction with the endocytic machinery.