V-1 is a 12 kDa protein containing 2.5 copies of the ankyrin repeat, which has been demonstrated to be required for protein-protein interactions. Recently we have for the first time reported that stable overexpression of V-1 enhances mRNA expression of catecholamine synthesizing enzymes in PC12D cells, and as a result, catecholamine production is upregulated. GTP cyclohydrolase I (GCH) is the enzyme in the first and rate-limiting step for the biosynthesis of tetrahydrobiopterin (BH4) which is an essential cofactor for tyrosine hydroxylase. In the present study, to examine further the function of V-1 in control of the BH4 biosynthesis, we assayed BH4 content and GCH enzyme activity in V-1-overexpressing PC12D cell clones. It was shown that both BH4 content and GCH enzyme activity were increased in V-1-verexpressing PC12D cell clones. It was also revealed that V-1-overexpression caused augmentation of both the GCH protein and mRNA expression and the cAMP-responsive element (CRE) dependent transcription. Furthermore, promoter analysis showed an increased activity in the construct with 150 bp of promoter region of the human GCH gene in the V-1-overexpressing clones. These results suggest that V-1 promotes GCH gene expression via a CRE-dependent transcription to positively control the BH4 biosynthesis in catecholaminergic cells.