Abstract—Tetrahydrobiopterin (BH₄) is an essential cofactor required for enzymatic activity of endothelial NO synthase. Recently, it has been shown that vascular protective effects of erythropoietin (EPO) are dependent on activation of endothelial NO synthase. Therefore, our objective was to characterize the effect of EPO on the biosynthesis of BH₄ in the vascular wall. Incubation of isolated C57BL/6J mouse aortas for 18 hours with recombinant human EPO (1 to 50 U/mL) caused a concentration-dependent increase in intracellular BH₄ levels and activity of GTP-cyclohydrolase I. Maximal biosynthesis of BH₄ was detected at therapeutic concentrations of 5 U/mL. Removal of the endothelium abolished EPO-induced biosynthesis of BH₄ demonstrating that the vascular endothelium is a major source of BH₄. Treatment with a selective phosphatidylinositol 3-kinase inhibitor wortmannin significantly reduced BH₄ biosynthesis stimulated by EPO. The stimulatory effect of EPO on vascular GTP-cyclohydrolase I activity, BH₄ production, and phosphorylation of endothelial NO synthase was also detected in vivo in mice treated with recombinant human EPO. These effects of EPO were abolished in protein kinase B/Akt1-deficient mice. In addition, EPO significantly increased systolic blood pressure and the number of circulating platelets in Akt1-deficient mice. Our results demonstrate that EPO stimulates biosynthesis of BH₄ in vascular endothelium and that the increase in BH₄ levels is caused by de novo biosynthesis of BH₄ via the phosphatidylinositol 3-kinase/Akt1 pathway. This effect is most likely designed to provide optimal intracellular concentration of the cofactor necessary for EPO-induced elevation of endothelial NO synthase activity.