Three C-terminal variants of the human norepinephrine transporter (hNET) are known: the wild-type hNET in which exon 14 encodes the last seven amino acids and two variants with either three or 18 amino acids encoded by an alternatively spliced exon 15. In transfected HEK293 cells we compared by means of [(3)H]norepinephrine ([(3)H]NE) uptake and [(3)H]nisoxetine ([(3)H]NIS) binding the functional properties of the wild-type hNET with those of the more abundant long splice variant containing exon 15 (hNET-Ex15L) and of two artificial hNET mutants lacking either the last three (hNET-Ex14-4) or all seven (hNET-Ex14-0) C-terminal amino acids of exon 14. No differences among the NET isoforms were observed concerning the K(m) for uptake of NE and the K(D) for binding of NIS. However, compared with the wild-type hNET, the three isoforms (hNET-Ex15L, hNET-Ex14-4 and hNET-Ex14-0) showed a pronounced decrease in V(max) of [(3)H]NE uptake and B(max) of [(3)H]NIS binding which correlated with strongly reduced surface expression of the transporter isoforms. The decrease in surface expression of the hNET isoforms is probably a consequence of the lack of the three amino acids leucine, alanine and isoleucine at the C-terminal end which may represent a motif facilitating cell surface expression of the hNET. Expression of hNET-Ex15L exerted a dominant negative effect on plasma membrane expression of the wild-type hNET and thus may represent a novel mechanism for regulation of noradrenergic neurotransmission.