The rat norepinephrine transporter (rNET) cDNA from the PC12 pheochromocytoma cell line has been cloned by RT-PCR and characterized. The cDNA encodes an integral membrane protein consisting of 617 amino acids which contains twelve putative transmembrane domains, two potential N-glycosylation sites, two potential phosphorylation sites for protein kinase C and one phosphorylation site for casein kinase II. The nucleotide and deduced amino acid sequence shows a high level of homology to the human and the bovine norepinephrine transporter and less homology to the rat dopamine transporter (rDAT). Heterologous expression of rNET in HEK293 cells revealed that uptake of [3H]norepinephrine is sodium- and chloride-dependent and highly sensitive to the selective norepinephrine transporter inhibitors desipramine and nisoxetine. The cloned rNET cDNA provides the opportunity to investigate this transporter in heterologous expression systems and adds a new member to the family of sodium- and chloride-dependent neurotransmitter transporters.