Purpose: Neovascularisation is a serious complication of several retinal diseases including diabetic retinopathy and retinopathy of prematurity. In these diseases an oxidative insult damages the endothelium leading to vascular closure, tissue ischaemia and the eventual abnormal neovascular response that characterizes these diseases. Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) plays an important role in maintaining vascular integrity, normal eNOS function, however is dependent upon the cofactor tetrahydrobiopterin (BH4) whose deficiency plays a pivotal role in the reduced NO bioavailability observed in diabetic vascular disease. We have previously shown that eNOS is dysfunctional in hyperoxia-induced vascular damage in a model of oxygen induced retinopathy (OIR). Thus, here our aim was to investigate the effect of hyperoxia-induced vascular closure in BH4 deficient mice (hph-1). Methods: BH4 levels in retinas, aortas and lung samples were analyzed by HPLC analysis. Normal vascular development was confirmed by lectin staining of retinas from postnatal day 7 (P7) animals. For OIR, animals were exposed to 75% oxygen for 5 days from P7 to P12 and returned to room air. Eyes were collected at various time points between P12 and P17 and vascular preservation and ischaemia-induced growth quantified by B simplicifolia isolectin staining of retinal flat mounts. Avascular, normal vascular and neovascular areas were quantified using image analysis software. NO production was quantified by NOS activity assay in the presence and absence of BH4. In situ superoxide and peroxynitrite were quantified by DHE, DCF and nitrotyrosine immunoreactivity. Results: BH4 levels in hph-1 retinal samples were significantly lower than wild-type litter mate controls and comparable to levels observed in aorta and lung samples. All BH4 levels were further reduced following in vivo hyperoxia treatment. Surprisingly, despite evidence of reduced retinal BH4 levels, hph-1 animals were partially protected from hyperoxia-induced damage which correlated with reduced reactive oxygen and nitrogen species production. In contrast, in response to ischaemia hph-1 animals displayed a significantly reduced neovascular response. Conclusions: Our results show that BH4 deficient hph-1 animals are protected from hyperoxia-induced vasodegeneration. However, the attenuated angiogenic drive observed in these animals suggests a reduced ability to respond to ischaemic insult.