Monoamine neurotransmitter transporters for norepinephrine (NE), dopamine and serotonin are important targets for antidepressants and analgesics. The conopeptide chi-MrIA is a noncompetitive and highly selective inhibitor of the NE transporter (NET) and is being developed as a novel intrathecal analgesic. We used site-directed mutagenesis to generate a suite of mutated transporters to identify two amino acids (Leu(469) and Glu(382)) that affected the affinity of chi-MrIA to inhibit [(3)H]NE uptake through human NET. Residues that increased the K(d) of a tricyclic antidepressant (nisoxetine) were also identified (Phe(207), Ser(225), His(296), Thr(381), and Asp(473)). Phe(207), Ser(225), His(296), and Thr(381) also affected the rate of NE transport without affecting NE K(m). In a new model of NET constructed from the bLeuT crystal structure, chi-MrIA-interacting residues were located at the mouth of the transporter near residues affecting the binding of small molecule inhibitors.