We recently described that hydrogen peroxide (H2O2) stimulates the synthesis of tetrahydrobiopterin (BH4) through the induction of the rate-limiting enzyme GTP-cyclohydrolase I (GTPCH), and increases tetrahydrobiopterin content in vascular endothelial cells. Tetrahydrobiopterin is easily oxidized by peroxynitrite (ONOO-), but not by hydrogen peroxide. The aim of this study was to determine the effect of hydroxyl radical and peroxynitrite, which are both toxic biological oxidants, on tetrahydrobiopterin synthesis and the regulation of its content in vascular endothelial cells. In the cell-free assay system, tetrahydrobiopterin was rapidly oxidized by the hydroxyl radical and peroxynitrite, but not by hydrogen peroxide. However, the addition of not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite to vascular endothelial cells transiently decreased tetrahydrobiopterin content, and then markedly increased its content. Interestingly, total biopterin content was also decreased by early treatment with oxidants. Moreover, oxidants induced the expression of GTP-cyclohydrolase I, and the increase of the tetrahydrobiopterin content was blocked by the treatment with GTP-cyclohydrolase I inhibitor. Both the hydrogen peroxide- and peroxynitrite-induced increases in tetrahydrobiopterin content and findings suggest that not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite stimulates tetrahydrobiopterin synthesis through GTP-cyclohydrolase I expression, and that the hydroxyl radical plays a central role in the stimulation of tetrahydrobiopterin synthesis. Moreover, the transient decrease in BH4 to tetrahydrobiopterin.