Hypothermia is the only neuroprotective therapy proven to be effective in stroke management. However, the therapeutic window of mild hypothermia has not been precisely elucidated. Therefore, the aim of this study is to optimize the time window of mild hypothermia in the endothelin-1 rat model for focal cerebral ischemia. In this model endothelin-1 is infused near the middle cerebral artery of male Wistar rats. We compared the neuroprotective effects of hypothermia initiated 60 minutes after the ischemic insult with hypothermia initiated after 20 minutes. The effects on the neurological outcome, infarct size and apoptosis were evaluated. Brain temperature was kept either under normothermic (37°C) or hypothermic conditions (33°C) for 2 hours starting with a delay of 20 or 60 minutes after the ischemic insult. Behavioural changes were scored according to a neurological deficit score, 24 hours after the insult and then the rats were sacrificed. Hypothermia, initiated 20 minutes after the onset of the insult, improved neurological outcome compared to normothermic rats. Hypothermia, initiated after 60 minutes, also improved neurological outcome although less pronounced. Infarct size was determined, using a cresylviolet staining. When hypothermia was initiated 20 minutes after the insult, infarct volume was reduced by half [normothermic (51.7mm³ 2.1mm³, mean SEM, n=10); hypothermic (25.7mm³ 2.0mm³, mean SEM, n=6) (Mann-Whitney, p<0.007)]. Hypothermia, initiated 60 minutes after the ischemic insult, also significantly reduced the infarct volume (31.4mm³ 0.9mm³, mean SEM, n=5). The present findings indicate that hypothermia remains neuroprotective even when the initiation of the treatment is delayed.