Transporters are important mediators of specific cellular uptake and thus for effects, but also side effects, metabolism and excretion of many drugs. In particular, cellular uptake of the tyrosine kinase inhibitor (TKI) imatinib and the cytostatic cisplatin seems to be critically mediated by organic cation transporters (OCT). Three subtypes of OCT exist (OCT1, OCT2 and OCT3) with specific organ and species dependent expression. In this work, we have compared the interaction of several TKIs and of platin derivatives with OCT from humans and, to add translational relevance, from mice cloned into human embryonic kidney (HEK) cells. The interaction of these drugs with OCT was examined fluorimetrically with the cation 4-(4-(dimethyl-amino)styril)-methylpyridinium iodide (ASP, 1 µM) as substrate. All TKI tested and cisplatin interacted with the ASP uptake by OCT. The IC50 differed both between the paralogs within a species and also between human and murine orthologs. Oxaliplatin did not interact with ASP uptake in any OCT. For this reason, we compared the toxicity of oxaliplatin in cells expressing or not hOCT2 by a colorimetric cell viability assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Incubation of hOCT2 expressing cells with oxaliplatin resulted in a higher cell toxicity than in cells without the transporter. In conclusion, our data show that TKI and cisplatin interact with OCT with different apparent affinities depending on the OCT subtype. Moreover, the interaction strength can be different between mouse and human OCT, indicating that translational studies should be interpreted with caution. As evident in the case of oxaliplatin, a drug can be unable to interact with the ASP transport mediated by OCT, but the transporters seem to be of critical importance for the development of drug-associated toxicities, suggesting that OCT have a broad binding site with different interaction domains for diverse substances.