Microglia contribute to the inflammatory response after ischemic stroke. Mild hypothermia (33°C) is a promising neuroprotective strategy and this study investigated whether such neuroprotection in the endothelin-1 (Et-1) rat model for transient focal cerebral ischemia, is associated with changes in gliosis. Therefore, Et-1 was infused near the middle cerebral artery in male Wistar rats which were subjected to 2 hours of mild hypothermia, started 20 minutes after Et-1 injection. They were compared with normothermic rats (37°C). Besides assessing functional outcome and infarct volume, the activation of microglia and astrocytes was evaluated using immunohistochemistry for CD-68 and glial fibrillary acidic protein (GFAP) respectively, both at the level of the core (striatum) and the penumbra (cortex). All parameters were determined up to one week after the insult. Et-1 administration caused neurological deficit and a reproducible infarct size. Both parameters were significantly reduced by hypothermia. There was a gradual increase in CD-68 expression up to 1 week after stroke onset. Hypothermia resulted in an attenuated expression of CD-68 compared to normothermic rats 8, 24 hours and 1 week after the insult. Surprisingly, at 3 days after the insult, hypothermia resulted in a 5-fold increase of CD-68 expression in both the core and the penumbra. GFAP expression peaked 1 day after the insult, after which it gradually decreased. Hypothermia significantly attenuated this response both in the core and the penumbra at 24 and 72 hours after the insult, but had no effect on the expression of GFAP at other time points. These data suggest that the beneficial effects of hypothermia after stroke on infarct volume and functional outcome may, at least partly, be mediated by inhibition of astrogliosis and microglial activation.