The goal of this study was to compare the substrate and inhibition profiles of the human renal organic cation transporter (hOCT2) and the hepatic hOCT1 to determine whether these transporters are functionally distinct. We examined the interactions of n-tetraalkylammonium (nTAA) compounds and biguanides (metformin, phenformin) with hOCT2 and compared results with previous findings for hOCT1. Substantial differences were found in the interactions of nTAAs with hOCT1 and hOCT2: for example, tetramethylammonium (TMA) had an 82-fold higher affinity for hOCT2 than hOCT1, while tetrabutylammonium (TBA) had a 4-fold higher affinity for hOCT1 than hOCT2. Trans-stimulation studies revealed that 3H-1-methyl-4-phenylpyridinium (MPP+) uptake by hOCT2 was trans-stimulated only by TMA and trans-inhibited by tetrapropylammonium (TPrA) and TBA, whereas hOCT1-mediated 3H-MPP+ uptake was trans-stimulated by tetraethylammonium (TEA) and TPrA. In contrast, the biguanides metformin and phenformin interacted similarly with both transporters—they inhibited 3H-cimetidine transport mediated by hOCT1 and hOCT2, trans-stimulated 3H-MPP+ influx for both, and metformin induced currents in hOCT2-expressing oocytes (providing evidence it is a hOCT2 substrate). This study demonstrates compound-dependent differences in the specificities of hOCT1 and hOCT2: hOCT2 (predominant in kidney) prefers smaller hydrophilic substrates, while hOCT1 (predominant in liver) interacts with larger, more hydrophobic compounds. These differences may contribute to organ-specific elimination of drugs and suggest significant distinctions in the binding sites of these paralogous transporters.