The dopamine transporter (DAT) is a presynaptic plasma membrane protein responsible for the termination of dopaminergic neurotransmission in the central nervous system. While most studies have focused on structure/function analysis, much less information is available regarding the assembly and the trafficking of this protein. To address this problem, we performed a mutational analysis of the DAT protein, combined with biochemical, immunological, and functional approaches. In mammalian cells co-expressing differentially tagged DAT molecules, HA-tagged DAT co-purified with 6His-tagged DAT demonstrating a physical interaction between transporter proteins. Evidence for the functional oligomerization of DAT was obtained using dominant-negative mutants of DAT. Two loss-of-function mutant transporters (Y335A and D79G) that were targeted to the cell surface inhibited wild-type DAT uptake activity without affecting the membrane targeting of the wildtype transporter. Moreover, non-functional amino and carboxyl termini-truncated mutants of DAT inhibited wild-type DAT function by interfering with the normal processing of the wild-type transporter to the cell membrane. Mutations in the leucine repeat of the second transmembrane domain of the transporter could eliminate the dominant-negative effect of all these mutants. In addition, a small fragment comprising the first two transmembrane domains of DAT inhibited wild-type transporter function but not when the leucine repeat motif was mutated. Taken together, our results suggest that the assembly of DAT monomers plays a critical role in the expression and function of the transporter. The dopamine transporter (DAT)1 belongs to a large family of Na ϩ /Cl Ϫ -dependent plasma membrane transporters that also includes the closely related norepinephrine and serotonin transporters (NET and SERT, respectively), and carriers for GABA, glycine, proline, taurine, and betaine. In the central nervous system, DAT mediates the re-uptake of released dopamine (DA) from the synaptic cleft back into the nerve terminal for subsequent storage and release. Pharmacological and genetic studies highlight the DAT-mediated re-uptake process as the main mechanism for the termination of dopamine neurotransmission (1). In addition, DAT represents the main target site for commonly abused drugs such as cocaine and amphetamine as well as some therapeutic agents used in the management of affective disorders (2). Hydrophobicity analysis of their deduced amino acid sequence reveals that Na ϩ /Cl Ϫ -dependent plasma membrane neurotransmitter transporters are proteins containing twelve transmembrane domains (TMs) with both the amino and the carboxyl termini located on the intracellular side of the membrane. This topological arrangement has been confirmed for several members of the family, including DAT (3). Since the molecular cloning of this transporter gene family, a great deal of information has been accumulated concerning the relationship between the structure and function of this class of proteins (4). Studies...