Tetrahydrobiopterin (BH4) is an obligatory cofactor and regulator of nitric oxide synthases (NOS). We evaluated the biosynthesis of BH4 in human umbilical vein smooth muscle cells (HUVSMC). Trace amounts of BH4 were found intra- and extracellularly in untreated cells. When HUVSMC were activated by individual inflammatory stimuli (IL-1beta, TNFalpha, IFNgamma or LPS), both intra- and extracellular levels of BH4 increased significantly, with TNFalpha being the most potent single stimulus. Combined inflammatory cytokines synergized in the induction of an up to 600-fold increase of BH4 synthesis. Addition of LPS to the cytokine mixture led to a further increase of BH4 synthesis. Neopterin, a product of the first intermediate in BH4 biosynthesis, was also raised, but to a much lesser extent. The increase of BH4 synthesis was paralleled by an enhanced expression of isoform-1 (the only isoform coding for the active enzyme) of GTP cyclohydrolase I in cytokine treated cells. Our results show for the first time that BH4 biosynthesis is strongly induced by combinations of inflammatory stimuli in HUVSMC. The importance of BH4-dependent NO synthesis in HUVSMC needs, however, additional detailed studies.