Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step of catecholamine synthesis and its expression is necessary for neurotransmitter specification of all catecholaminergic neurons, while dopamine beta-hydroxylase (DBH) is essential for the noradrenergic phenotype. In the present study, we show that Nurr1, an orphan nuclear receptor critical for dopaminergic (DA) neuron development, directly transactivates the promoter activity of the TH gene in a cell type-dependent manner, while it does not regulate the DBH promoter. Consistent with these results, only the TH promoter contains multiple sequence motifs homologous to the known Nurr1-binding motif, NBRE. TH promoter deletional analysis indicates that < 1.0 kb upstream sequences, encompassing three NBRE-like motifs (i.e. NL1, NL2 and NL3) are mostly responsible for the effects of Nurr1. Among these potential motifs, site-directed mutational analysis showed that NL1, residing from - 35 to - 28 bp, was most critical for mediating the transactivation by Nurr1. Strikingly, however, both DNase I footprinting and electrophoretic mobility shift assays showed that NL3, but not NL1 or NL2, has high binding affinity to Nurr1. To determine whether the proximity of these motifs may be important for transactivation by Nurr1 in the transient transfection assay, we generated reporter gene constructs in which NL3 is immediately proximal to the TATA box. Indeed, NL3 was more efficient in this position than NL1 or NL2 for mediating the transactivation by Nurr1. Our results suggest that Nurr1 may play a direct role for specification of DA neurotransmitter identity by activating TH gene transcription in a cell context-dependent manner.