Activation of the hypoxia inducible factors (HIF) pathway by inhibition of the HIF prolyl-4-hydroxylase enzymes (PHD) confers protection against ischemia/reperfusion injury (Sridharan V, et al. 2007). We investigated the precise function of each PHDs (1-3) in ischaemic stroke by using mice with each isoform genetically suppressed and by applying a novel specific PHD inhibitor – FG2216. Middle cerebral artery occlusion (MCAO)(45min ischaemia/24h reperfusion) was performed under anesthesia with 1.5% isoflurane in O2/N2O (1:3) on male, 8-12 week old PHD1-/-, PHD2+/- and PHD3-/- mice and their wild type (WT) littermates, as well as C57/B6 mice treated with FG2216. During the experiments, regional cerebral blood flow (rCBF) was recorded by a laser Doppler flowmetry. Behaviour was assessed at 24h after reperfusion with a common neuroscore. Infarct volumes, blood brain barrier (BBB) disruption, cerebral vascular density, reactive oxygen species (ROS) and apoptosis were then determined using histological and immunohistochemical techniques. When compared to their WT littermates, PHD2+/- mice had significantly more effective restoration of rCBF upon reperfusion, better functional outcomes (Neuroscores: 1.9±0.5 vs 4.3±0.5, p<0.01) and higher activity rates at 24h after MCAO, there were significantly fewer apoptotic cells in the penumbra and less BBB disruption, with a trend towards reduced infarct volume (26.7±6.7% vs 40±5.5%, p=0.15) but no difference in ROS formation; PHD3-/- mice had impaired rCBF upon early reperfusion but comparable functional outcomes; PHD1-/- mice did not show any significant changes following MCAO. When receiving FG2216 one day before the MCAO, C57/B6 mice had better neuroscores and smaller infarct volumes than those receiving the vehicle (Neuroscores: 2.0±0.6 vs 4.5±0.3; Infarct volumes: 27.0±8.1% vs 47.4±5.3%, both p<0.05). No significant difference was seen in mice if receiving FG2216 immediately before the MCAO. Genetic inhibition of PHD enzymes produces different effects on outcome after transient cerebral ischemia. Notably, partially knock-out PHD2 confers neuroprotection for cerebral ischaemia. These effects are seen following lifelong PHD deficiency and thus reflect a composite of developmental changes, affecting features such as vessel density and overall sympathetic tone. While Fg2216 induces acute HIF activation amplifying the hypoxic stimuli inherent in this ischaemic/reperfusion model which could contribute to the observed protection. HIF activation upon ischaemic injury is swift, but the HIF transcription is delayed (Sharp FR, et al. 2004). Our study needs to be considered in optimizing therapeutic effects of PHD inhibitors, particularly when isoform specific inhibitors become available.