Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthase (NOS), thus BH4 might play a key role for maintaining endothelial functions. Here we examined whether hypoxia affects intracellular BH4 levels under inflammatory cytokine-stimulated human umbilical vein endothelial cells (HUVEC). IFN-gamma and TNF-alpha (each 15 ng/mL) markedly increased BH4 levels after incubation for 24h. Hypoxia (3%, O₂) significantly caused further increase of the BH4 levels than those in normoxia. Hypoxia also caused decreasing levels of BH2, which inhibits NOS activity and causes production of superoxide anion. Contrary to the increased BH4 levels, hypoxia reduced mRNA expression of GTP cyclohydrolase 1 (GCH1), the rate limiting enzyme in BH4 biosynthesis. As to the increased intracellular BH4 levels, we considered two possibilities for the reason: (1) elevated activity of dihydrofolate reductase, which is able to reduce BH2 to BH4; (2) turn back of once released BH4 into cells by autocrine or paracrine manner, since degradation of BH4 might less occur under hypoxic condition. The decrease of GCH1 mRNA expression suggested the presence of a feedback mechanism in BH4 biosynthesis other than the feedback mediated by GTP cyclohydrolase 1 feedback regulatory protein.