The organic cation transporter 2 (OCT2) provides an important pathway for the uptake of cationic compounds in the kidney, which is the essential step in their elimination from the organism. Although many drugs have been identified which interact with human OCT2, structural elements required for an interaction with OCT2 are not well defined. To address this issue, HEK293 cells stably expressing human OCT2 were generated. IC₅₀ values of commonly used drugs for inhibition of [³H]MPP⁺ uptake were determined and correlated with physicochemical descriptors. We found only a significant correlation between the topological polar surface area (TPSA) and IC₅₀ values (r = 0.71, p < 0.0001). Structural alignment of most potent inhibitor drugs of OCT2-mediated MPP⁺ uptake was used to construct a two-point pharmacophore consisting of an ion-pair interaction site and a hydrophobic aromatic site separated by 5.0 Å. Taken together, our data identify structural determinants for inhibitor interactions with OCT2.