Strategies to pharmacologically modulate pathways that stimulate endogenous nitric oxide (NO) bioavailability have generated considerable interest for the treatment of cardiovascular disease. Tetrahydrobiopterin (BH4) is an essential cofactor for the synthesis of nitric oxide (NO). Clinical studies have shown that intravenous administration of BH4 improves vascular function in cardiovascular disease patients, however oral BH4 appears to lack efficacy, possibly due to oxidative inactivation during absorption. BH4 is synthesised from GTP with GTP-cyclohydrolase 1 (GCH1) catalysing the first and rate limiting step of the reaction. The amino acid L-phenylalanine (L-Phe) can activate GCH-1 when it is complexed to an allosteric regulatory protein (GFRP); indeed oral administration of L-Phe increases plasma biopterin (a marker of BH4 levels) in humans. The aim of this study was to investigate the effects of exogenous L-Phe administration on BH4 levels and vascular function in wild type mice and spontaneously hypertensive rats (SHR). Wild type mice were administered with an oral L-Phe challenge (100 mg/Kg) and BH4 and NO levels measured in multiple tissues. Concentrations of nitrite, BH4 and catecholamines in tissue/plasma and superoxide levels in aorta, were measured in male SHRs and wild type normotensive rats (WKY). The effect of L-Phe on endothelium dependent vascular relaxation to acetylcholine was assessed functionally using aortic and mesenteric vessels from SHR and WKY. All tissues were collected after animals were exsanguinated under terminal anaesthesia (2% isofluorane).Oral L-Phe challenge caused a significant and temporal rise in BH4 tissue levels in wild type mice, an effect that was pronounced in aorta (n=8). Consistent with their hypertensive pathology, BH4 and nitrite levels were lower whilst catecholamines and superoxide levels were elevated in SHR compared to WKY (n=18). SHR demonstrated endothelial dysfunction compared to WKY basally in aortic and mesenteric tissues [EC50 aorta (µM): SHR0.21±0.004 vs WKY0.08±0.0005; n=12] [EC50 mesenteric (µM): SHR0.03±0.0001 vs WKY0.01±0.003; n=6]. Incubation with 500 µM L-Phe significantly improved ACh induced vasorelaxation in SHR whilst having no effect in WKY (EC50 shifted to 0.038±0.0007 µM and 0.014±0.002 µM in SHR aorta and mesenteric respectively; n=6). The L-Phe effect was found to be endothelium-dependent. L-Phe was used as a tool to investigate the potential to activate GCH1 and enhance vascular function. Consistent with our hypothesis , oral L-Phe enhanced aortic BH4 in wild type mice and improved vascular relaxations in SHR ex vivo. Small molecules that mimic this allosteric activation of GCH1 represent a potential novel therapy for the treatment of endothelial dysfunction, circumventing the problems with administering BH4 orally.