Aims: Human organic cation transporters (hOCT1-3) are transmembranal proteins involved in the transport of endogenous and exogenous organic cations. Because of their localisation, these transporters play a primary role in the extraneuronal homeostasis of circulating catecholamines (hOCT3) and in the hepatic and renal secretion and reabsorption of drugs (hOCT1 and hOCT2). Since interactions of hOCTs with other proteins are important for their function and regulation, identification of such interaction partners of hOCTs and determination of their function is of relevance. Methods: To identify unknown interaction partners of hOCT1-3 in vivo, a membrane-based Split-Ubiquitin-Yeast-Two-Hybrid (SU-YTH) technique was used. Interactions were confirmed by pull-down assays, FRET analysis and immunohistochemistry. The fluorescent organic cation 4-4-dimethylamino-styryl-N-methylpyridinium (ASP) was used as a model cation to monitor transporter function in HEK293-cells. Results: Several proteins directly interacting with hOCTs were identified by the SU-YTH. We have focused on two proteins, which interact with all three isoforms of hOCTs: LAPTM4A, a transmembrane protein associated with lysosomes, and CD63, a tetraspanin located in late endosomes. The interactions were confirmed by pull-down experiments and/or FRET analysis. Overexpression of CD63 and LAPTM4A significantly decreased ASP uptake in HEK293 cells stably transfected with hOCT2 or hOCT3, suggesting a negative regulation of hOCT mediated transport. Downregulation of endogenous CD63 expression by siRNA increased ASP transport. Conclusion: We have identified two proteins of the endosomal pathway that interact with hOCT1-3. This interaction seems to regulate the function of OCTs by influencing their trafficking to/from the cell membrane. Therefore, also the intracellular sorting machinery could have significant physiological and pharmacological implications by regulating the number of active OCTs on the plasma membrane.