AbstractTetrahydrobiopterin is an essential cofactor for the phenylalanine, tyrosine and tryptophan hydroxylases, and the family of nitric oxide synthases. The initial and rate‐limiting enzyme in the biosynthesis of tetrahydrobiopterin is GTP cyclohydrolase I. The proximal promoter of the human GTP cyclohydrolase I gene contains the sequence motif 5′‐TGACGCGA‐3′, resembling a cAMP response element (CRE). The objective of this study was to analyze the regulation of GTP cyclohydrolase I gene transcription by basic region leucine zipper (bZIP) transcription factors. A constitutively active mutant of the cAMP response element binding (CREB) protein strongly stimulated GTP cyclohydrolase I promoter activity, indicating that the CRE in the context of the GTP cyclohydrolase I gene is functional. Likewise, GTP cyclohydrolase I promoter/luciferase gene transcription was stimulated following nuclear expression of the catalytic subunit of cAMP‐dependent protein kinase. Constitutively active mutants of activating transcription factor 2 (ATF2) and c‐Jun additionally stimulated GTP cyclohydrolase I promoter activity, but to a lesser extent than the constitutively active CREB mutant. The fact that stress‐activated protein kinases target the GTP cyclohydrolase I gene was corroborated by expression experiments involving p38 and MEKK1 protein kinases. We conclude that signaling pathways involving either the cAMP‐dependent protein kinase or stress‐activated protein kinases converge to the GTP cyclohydrolase I gene. Hence, enzymatic reactions that require tetrahydrobiopterin as cofactor are therefore indirectly controlled by signaling cascades involving the signal‐responsive transcription factors CREB, c‐Jun, and ATF2. J. Cell. Biochem. © 2005 Wiley‐Liss, Inc.