Therapeutic hypothermia has shown neuroprotective promise, but whether it can be used to improve outcome in stroke has yet to be determined in patients. Recombinant tissue plasminogen activator (rt-PA) is only given to a minority of patients with acute ischemic stroke, and is not without risk, namely significant brain hemorrhage. We explored whether mild hypothermia, in combination with rt-PA, influences the safety of rt-PA. Mice were subjected to middle cerebral artery occlusion (MCAO) using a filament model, followed by 24 hours reperfusion. Two paradigms were studied. In the first paradigm, cooling and rt-PA treatment began at the same time upon reperfusion, whereas in the second paradigm, cooling began soon after ischemia onset, and rt-PA began after re-warming and upon reperfusion. Experimental groups included: tPA treatment at normothermia (37 degrees C), rt-PA treatment at hypothermia (33 degrees C), no rt-PA at normothermia, and no rt-PA treatment at hypothermia. Infarct size, neurological deficit scores, blood brain barrier (BBB) permeability, brain hemorrhage, and expression of endogenous tissue plasminogen activator (tPA) and its inhibitor, plasminogen activator inhibitor (PAI-1) were assessed. For both paradigms, hypothermia reduced infarct size and neurological deficits compared to normothermia, regardless of whether rt-PA was given. rt-PA treatment increased brain hemorrhage and BBB disruption compared to normothermia, and this was prevented by cooling. However, mortality was higher when rt-PA and cooling were administered at the same time, beginning 1-2 hours post MCAO. Endogenous tPA expression was reduced in hypothermic mice, whereas PAI-1 levels were unchanged by cooling. In the setting of rt-PA treatment, hypothermia reduces brain hemorrhage, and BBB disruption, suggesting that combination therapy with mild hypothermia and rt-PA appears safe.