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When assayed in parallel using transfected mammalian cells, human and rat serotonin transporters (SERTs) exhibit consistent differences in potency for tricyclic antidepressants but not for 5-hydroxytryptamine, cocaine, or nontricyclic serotonin transporter-selective reuptake inhibitors. Previously, using chimeric proteins, we determined that domains or residues distal to transmembrane domain 11 (amino acid 531) dictate the increased sensitivity of human SERT to imipramine. Using an additional chimera and site-directed mutagenesis, we have determined that a single amino acid, F586, is responsible for increased sensitivity to imipramine, desipramine, and nortriptyline. Thus, mutation of wild-type rat SERT (V586) to the human SERT identity F586, but no other divergent amino acids between human and rat SERTs, selectively increased tricyclic antidepressant potency. A reciprocal reduction in potency was observed when human SERT F586 was converted to the cognate rat SERT residue (V586). Interactions with other SERT antagonists, including paroxetine and cocaine, as well as the SERT substrates 5-hydroxytryptamine and d-amphetamine were unaffected by interconversion of this residue. Phenylalanine conversion in the human norepinephrine transporter at the homologous position failed to alter tricyclic inhibition of catecholamine uptake, revealing a SERT-specific context for use of the aromatic side chain at this position. Additional constraints on aromaticity at rat SERT position 586 were revealed by conversion of rat SERT V586 to Y586, which failed to repllcate the effect of the F586 mutation. In addition, conversion to V586D, but not V586R, increased tricyclic potency to that of human SERT and additionally increased potency for cocaine but not paroxetine. These results implicate distal domains and a single residue in TMD 12 in the formation of high affinity SERT antagonist binding sites.