OCT are membrane proteins involved in the transport of endogenous (e.g. the biogenic amines dopamine and histamine) and of exogenous organic cations. OCT are highly expressed in excretory organs (kidney, liver) and also in the brain. For this reason, OCT have a crucial importance in modulating cerebral and renal dopaminergic activity and dopamine clearance from the blood. OCT determine also the efficacy and toxicities of drugs such as biguanides, platin derivatives, and tyrosine kinase inhibitors. OCT have been assigned to the SLC22A family. This family can be divided into various subgroups according to substrates and transport mechanisms. Three OCT subtypes (OCT1, OCT2, and OCT3) with specific organ distribution have been identified. In this work, we focused on human OCT2 (hOCT2), the renal subtype of OCT. Since one of the factors determining transporter function and regulation is the building of complexes with other proteins or with itself, we searched for interaction partners of OCT and investigated the functional role of such a complex. For these investigations, a mating-based split-ubiquitin-yeast-two-hybrid system (mbSUS), fluorescence resonance energy transfer (FRET), Western blot analysis, crosslinking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4-4-(dimethylamino)styryl)-N-methylpyridinium (ASP) were applied to HEK293 cells stably transfected with hOCT2 and partly also in freshly isolated human proximal tubules. Since the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes and if so which part of the transporter is involved in the oligomerization. The role of cysteines for oligomerization and trafficking to the plasma membranes was investigated in cysteine mutants of hOCT2. The mbSUS, FRET, and Western blot analysis shows that hOCT2 forms oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutations of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for a correct trafficking to the plasma membrane and for oligomerization. Other than with itself, hOCT2 also interacts with the lysosomal associated protein transmembrane 4 alpha (LAPTM4A) as demonstrated by mbSUS, FRET, and pull-down experiments. LAPTM4A is an intracellular localized membrane protein associated to lysosomes and late endosomes. Functionally, overexpression of LAPTM4A significantly decreased ASP uptake in HEK293 cells stably transfected with hOCT2, suggesting a negative regulation of hOCT2 mediated transport. Furthermore, overexpression of LAPTM4A led to a significantly decreased hOCT2 plasma membrane expression in surface biotinylation experiments. Significant expression of LAPTM4A in human kidney was demonstrated by immunoblotting and immunofluorescence. The interaction hOCT2-LAPTM4A may give origin to a tandem structure, where organic cations, binding at the plasma membrane to hOCT2, are immediately directed by LAPTM4A to lysosomes, where they accumulate. Alternatively, the interaction leads to recruitment of OCT in an endocytotic compartment, where the complex hOCT2-LAPTM4A is digested. Another direct interaction partner identified by mbSUS and confirmed by pull-down and also by FRET-FACS analysis is the tetraspanin CD63. Tetraspanin CD63 is a ubiquitously expressed protein of the endosomal system which also moves to the cell surface. Overexpression of CD63 significantly decreased organic cation transport in HEK293 cells stably transfected with hOCT2, while its down-regulation by siRNA stimulated it, suggesting a negative regulation of hOCT2 mediated transport by CD63. Furthermore, overexpression of CD63 significantly decreased hOCT2 plasma membrane localization in surface biotinylation experiments. Total interne reflection resonance experiments showed CD63-hOCT2 interaction in the plasma membrane but stronger in vesicular compartments. Expression and colocalization of CD63 with hOCT2 was demonstrated in human kidneys by immunohistochemistry. As functional consequences of CD63 knockout in mice we observed that hOCT2 lost its specific basolateral localization in the kidney, appearing to be widespread distributed in the cell with a significantly decreased transport rate. In conclusion, CD63 regulates in a physiologically relevant fashion the function of hOCT2. We speculate that CD63 influences its trafficking to/from the cell membrane and processing via the intracellular sorting machinery and that this processing depends on polarization behaviour of the cells. Supported by the Deutsche Forschungsgemeinschaft (DFG CI 107/4-1 to 2).