Transcription factor NF-kappaB is associated with inflammatory response and cell survival. Under inactive condition, NF-kappaB is sequestered in the cytoplasm by an anchor protein, inhibitor of NF-kappaB (IkappaB). NF-kappaB was shown to be activated during ischemic brain injury. In the present study we have investigated the role of NF-kappaB in ischemic brain injury using a recombinant adenovirus expressing a dominant negative form of IkappaB (Adv/IkappaBdn) to specifically inhibit NF-kappaB activation. Our data demonstrated that cortical injection of Adv/IkappaBdn significantly reduced ischemic brain injury following permanent occlusion of the middle cerebral artery (MCAO) in rats, showing 55% reduction (p<0.01, n=8) in total ischemic lesion or 80% reduction (p<0.001) in the cortical area with Adv/IkappaBdn expression. Similarly, Adv/IkappaBdn expression significantly decreased neurological deficits (37% reduction over controls, p<0.01, n=8). These data provide further evidence for the role of NF-kappaB/IkappaB in ischemic brain injury and suggest that inhibition of NF-kappaB is neuroprotective in focal stroke.