Serotonin (5-HT) biosynthesis depends on two rate-limiting tryptophan hydroxylases, of which Tph1 is expressed in peripheral organs and Tph2 in neurons. Because 5-HT is involved in pulmonary hypertension (PH), we investigated whether genetic variations in Tph1 and/or Tph2 affected PH development in mice. To examine the functional impact of peripheral Tph1 deficiency on hypoxic PH, we used Tph1-/- mice characterized by very low 5-HT synthesis rates and contents in the gut and lung, contrasting with increased 5-HT synthesis in the forebrain. With chronic hypoxia, 5-HT synthesis in the forebrain increased further. Hypoxic PH, right ventricular hypertrophy, and distal pulmonary artery muscularization were less severe (P<0.001) than in wild-type controls. The Tph inhibitor p-chlorophenylalanine (PCPA, 100 mg/kg/d) further improved these parameters. We then investigated whether mouse strains harboring the C1473G polymorphism of the Tph2 gene showed different PH phenotypes when exposed to hypoxia. C57Bl/6 and 129X1/SvJ mice homozygous for the 1473C allele exhibited higher forebrain Tph activity and greater severity of hypoxic PH than DBA/2 and BALB/cJ mice homozygous for the 1473G allele. Treatment with PCPA reduced PH in all groups and abolished the difference in PH severity across mouse strains. Hypoxia increased 5-HTP accumulation but decreased 5-HT contents in the forebrain and lung, suggesting accelerated 5-HT turnover during hypoxia. These results provide evidence that dysregulation of 5-HT synthesis is closely linked to the hypoxic PH phenotype in mice and that both Tph1 and Tph2 may contribute to PH development.