BACKGROUND AND PURPOSE: In 2 models of severe ischemic injury, we have evaluated the neuroprotective action of D-JNKI1, a cell-penetrating and protease-resistant peptide selectively inhibiting the c-Jun-N-terminal kinase (JNK). METHODS: Hippocampal slices from newborn rats were subjected to oxygen (5%) and glucose (1 mmol/L) deprivation for 30 minutes. Cell death was evaluated with propidium iodide, and the evoked potential responses were recorded in the CA1 region after stimulation in CA3. Male ICR-CD1 mice were subjected to permanent endoluminal "suture" middle cerebral artery occlusion (MCAo). The lesion size was determined after 24 hours by triphenyl-tetrazolium chloride staining, and neurological scores and rotarod treadmill performance were used to evaluate the neurological outcome. RESULTS: In vitro, D-JNKI administration 6 hours after oxygen glucose deprivation reduced cell death at 24 hours from 21%+/-8% (n=10) to 5%+/-3% (n=7, P<0.01). This protective effect was still seen at 48 hours, paralleled by an improved amplitude of the evoked potential response. In vivo in the mouse, D-JNKI1 administration 3 hours after ischemia significantly reduced the infarct volume from 162+/-27 mm(3) (n=14) to 85+/-27 mm(3) (n=9, P<0.001). The functional outcome was also improved. CONCLUSIONS: JNK inhibition prevents cell death induced by oxygen and glucose deprivation in hippocampal slice cultures in vitro and by permanent suture MCAo in vivo. D-JNKI1 is a powerful neuroprotectant in models of both mild and severe cerebral ischemia, with an extended therapeutic window. Further investigations are needed to identify the relevant JNK target(s) mediating ischemic neuronal death.