(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.Catecholamines, e.g. dopamine (DA), 1 norepinephrine (NE), and epinephrine, and serotonin (5-hydroxytryptamine; 5HT) are well known to be involved in many aspects of brain function, including mood, addiction, reward, and sleep. Alteration in the metabolism of these monoamines leads to abnormalities in behavior and neuropsychiatric states. For example, increased dopaminergic neurotransmission in the mesolimbic system induces hallucinations, illusions, and delusions, which are the major symptoms of schizophrenia (1). Depletion of monoamines may be the cause of depression, because drugs that can increase the level of monoamines and blockers of norepinephrine or serotonin uptake show therapeutic effects in patients with depression (2). Abnormal monoamine metabolism is also suspected to be involved in Tourette's syndrome, obsessive-compulsive disorder, Rett syndrome, and infantile autism (3, 4).Biosynthesis of catecholamines and serotonin is mainly regulated by the activity of the hydroxylation reaction catalyzed by tyrosine hydroxylase (TH) in catecholamine synthesis (5) and by tryptophan hydroxylase (TPH) in 5HT synthesis (6). The TH activity is tightly regulated by several factors (for review, see Ref. 7 and references therein). Short term regulation is achieved by phosphorylation of the enzyme and by feedback inhibition with the end products. Long term regulation is mainly governed at the transcriptional level.(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for both TH and TPH. Although many cofactors such as folic acid and cobalamin (vitamin B12) are required to be dietary supplemented, mammals have a de novo biosynthetic pathway for BH4. In this pathway, guanosine triphosphate (GTP) is converted to 7,8-dihydroneopterin triphosphate by GTP cyclohydrolase 1 (GCH), and 7,8-dihydroneopterin triphosphate is then converted to 6-pyruvoyltetrahydropterin by pyruvoyltetrahydropteri...