The rewarding effects and addiction potential of psychotimulants such as cocaine and amphetamine are the result of their molecular interaction with the dopamine transporter (DAT). DAT belongs to the SLC6 (solute carrier 6) gene family of Na+/Cl- dependent transporters and mediates rapid reuptake of dopamine from the synaptic cleft. The long-term goal of our research is to understand the molecular and cellular mechanisms governing the activity and availability of the DAT and related transporters in the synaptic membrane. By the combined use of molecular modeling, site-directed mutagenesis, cysteine reactivity assays and fluorescence techniques we are currently trying to define the structural basis for conformational transitions in the transport cycle as well as we aim at characterizing the structural basis for the action of cocaine, amphetamines and related psychostimulants at DAT. In parallel, we investigate how protein-protein interactions regulates DAT activity and DAT trafficking. We have shown that Ca2+/calmodulin dependent kinase IIa (CaMKIIa) binds the DAT C- terminus and that this binding facilitates phosphorylation of the DAT N-terminus, which in turn mediates dopamine efflux in response to amphetamine. Recently, we have also developed a series of fluorescently tagged cocaine analogues that have allowed visualization for the first time of natively expressed DAT and its trafficking properties in dopaminergic neurons. Finally, to investigate the role of C-terminal PDZ-domain interactions for regulation of DAT in native tissue, we have generated knock-in mice expressing a mutant DAT with perturbed ability to interact with PDZ-domain proteins. Our data suggest that PDZ domain interactions play a key role in regulating axonal and dendritic localization of the DAT protein, and thereby for overall regulation of dopaminergic signalling in the brain.