This study examined whether tetrahydrobiopterin (BH4) stimulates angiogenesis by measuring the formation of tube-like structures in vascular endothelial cells. Bovine aortic endothelial cells that were cultured between two layers of collagen type I gel formed tube-like networks. Addition of BH4 or sepiapterin, a precursor of BH4 synthesis, stimulated the formation of tube-like structures. The sepiapterin-stimulated tube formation was completely inhibited by co-treatment with N-acetylserotonin, an inhibitor of sepiapterin reductase. These findings show that BH4 stimulates in vitro angiogenesis in vascular endothelial cells.