The study examines the roles of the highly conserved MELAL and GQXXRXG motifs, located in the second transmembrane domain and the first intracellular loop of the human noradrenaline transporter (hNET). We have previously shown that this region does not directly participate in the NET substrate translocation pathway [Sucic, S., and Bryan-Lluka, L.J., 2005. Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis. J. Neurochem. 94, 1620-1630.], while the current report focuses on the importance of this region in determining other functional properties of the hNET. Mutation to cysteine of the wild-type residues was carried out by site-directed mutagenesis of hNET cDNA. The wild-type and mutant hNETs were expressed in transiently transfected COS-7 cells and the effects of these mutations were pharmacologically examined. The results indicate that the GQXXRXG motif is important for the binding of cocaine, but not antidepressants. The hN120C mutant caused an 11-fold increase in the binding affinity of cocaine, compared to the wild-type hNET, while hQ118C, hY119C, hR121C and hE122C showed smaller increases. Interestingly, the apparent affinities of cocaine for some of these mutants were either decreased or unchanged, contrasting with the effects observed from the binding studies. The hE113C mutant in the MELAL motif caused very marked (over 400-fold) reductions in the binding affinities of substrates, but had no effects on the binding affinities of cocaine or antidepressants. Overall, the MELAL and GQXXRXG motifs are important determinants of NET cell surface expression and substrate and inhibitor binding. The results further suggest that the binding sites for substrates, cocaine and antidepressants on the NET are distinct but overlapping.