The aim was to examine the functional importance in the norepinephrine transporter (NET) of (i) the phenylalanine residue at position 531 in transmembrane domain (TMD) 11 by mutating it to tyrosine in the rat (rF531Y) and human (hF531Y) NETs and (ii) the highly conserved tyrosine residues at positions 249 in TMD 4 of human NET (hNET) (mutated to alanine: hY249A) and 271 in TMD 5, by mutating to alanine (hY271A), phenylalanine (hY271F) and histidine (hY271H). The effects of the mutations on NET function were examined by expressing the mutant and wildtype NETs in COS-7 cells and measuring the K(m) and V(max) for uptake of the substrates, [3H]norepinephrine, [3H]MPP(+) and [3H]dopamine, the K(D) and B(max) for [3H]nisoxetine binding and the K(i) of the inhibitors, nisoxetine, desipramine and cocaine, for inhibition of [3H]norepinephrine uptake. The K(m) values of the substrates were lower for the mutants at amino acid 271 than hNET and unaffected for the other mutants, and each mutant had a significantly lower V(max) than NET for substrate uptake. The mutations at position 271 caused an increase in the K(i) or K(D) values of nisoxetine, desipramine and cocaine, but there were no effects for the other mutations. Hence, the 271 tyrosine residue in TMD 5 is an important determinant of NET function, with the mutants showing an increase in the apparent affinities of substrates and a decrease in the apparent affinities of inhibitors, but the 249 tyrosine and 531 phenylalanine residues do not have a major role in determining NET function.