Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470 and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO₂-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis et al., J Biol Chem 281: 35272-35280, 2006). Thus, it was hypothesized that the environmental contaminant Hg²⁺ (as HgCl₂) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [³H]TEA into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl₂, with an IC₅₀ of 3.9 ± 0.11 µM. Treatment with 10 µM HgCl₂ caused a 6-fold reduction in the maximal rate of TEA transport, but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg²⁺, a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl₂ than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl₂ effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg²⁺ with these residues causes reduced TEA transport activity.