Functional expression of norepinephrine transporter (NET) and its regulation were examined in rat pheochromocytoma cell line, PC12. Nerve growth factor (NGF) decreased [3H]-norepinephrine (NE) uptake in association with a decrease in NET mRNA levels. On the other hand, levels of tyrosine hydroxylase mRNA increased in PC12 cells treated with NGF for 4-24 h, while Oct-2 mRNA levels decreased at 4 h with NGF then recovered for 8-24 h in the presence of NGF. Both bFGF and EGF reduced [3H]NE uptake, although they failed to affect NET mRNA levels. To examine the NET transcriptional regulation, we identified the 5'-noncoding region of rat NET mRNA by the rapid amplification of cDNA end (RACE) method and cloned the 5'-flanking region of NET gene. The newly identified exon encodes the untranslated region of rat NET mRNA upstream of the known 5'-region including ATG start codon. Constructs having green fluorescent protein (GFP) as reporter were made with the cloned NET gene, and promoter activity was examined in CHO and SK-N-SH cells transiently transfected and in PC12 cells stably transfected with NET-GFP constructs. The results indicate that the 2.1 kb NET flanking region displays promoter activity and is responsible for the NGF-induced down-regulation of NET expression.