Cerebral stroke is one of the leading causes of adult morbidity within the UK. Oxidative stress is a major consequence of reperfusion injury, resulting in disruption of the blood-brain barrier and neuronal cell death. Activation of the redox-sensitive transcription factor NF-E2 related factor 2 (Nrf2) affords increased protection against stroke via the upregulation of antioxidant defence enzymes such as heme oxygenase-1 (HO-1) and NADPH:quinone oxidoreductase 1 (NQO1). Quantitative immunohistochemistry was employed to examine temporal and spatial distribution of Nrf2 content in contralateral and stroke-affected regions in ex vivo brain sections from male Sprague-Dawley rats (250-300g) subjected to 70 min middle cerebral artery occlusion (MCAo) and reperfusion injury (4, 24 and 72h). Rats were also pre-treated with the Nrf2 inducer sulforaphane (SFN, 5mg/kg i.p.) 1h prior to MCAo followed by 24h reperfusion injury. Coronal brain sections (10µm) were obtained from perfusion-fixed brains and incubated with an anti-Nrf2 primary and horseradish peroxidase conjugated secondary antibody. Core and peri-infarct regions were identified based on glial fibrillary acidic protein (GFAP) expression. Sections were reacted with 3,3′-diaminobenzedine (DAB) and H2O2, and initial rates of DAB polymer formation ((sx103)-1, mean ± S.E.M., n=3-5 animals per group) were measured as an index of Nrf2 concentration. Image processing was used to determine Nrf2 content in nuclear and cytoplasmic compartments. Moreover, murine bEnd.3 brain endothelial cells were treated with SFN (2.5µM) and time dependent expression of HO-1 and NQO1 assessed by immunoblotting. Nrf2 content was greater in cytoplasm compared to nucleus after 4h reperfusion, but was increased in nuclear compartments following 24h reperfusion injury. After 72h, nuclear levels of Nrf2 returned to levels observed at 4h. Total cellular Nrf2 content was greater after 24h reperfusion, with increased levels noted in peri-infarct (1.35±0.016) compared to stroke core (0.72±0.016, P<0.001) regions. Cellular Nrf2 content was also elevated in contralateral regions (0.38±0.005) of stroke versus naïve (0.27±0.004, P<0.001) animals. After 72h, cellular Nrf2 content decreased in stroke-affected regions, but remained elevated in peri-infarct cells. Notably, SFN pre-treatment significantly reduced cellular Nrf2 content in contralateral regions (0.18±0.002) and in peri-infarct (0.049±0.008) and core (0.23±0.005) regions of the stroke hemisphere after 24h reperfusion compared to untreated animals subjected to reperfusion injury (P<0.001). Furthermore, SFN induced a time dependent increase in HO-1 and NQO1 expression in bEnd.3 cells. We conclude that SFN pre-treatment activates the Nrf2 defence pathway, affording protection of the brain against oxidative injury in stroke.