The brain endothelium constitutes a barrier to the passive movement of substances from the blood into the cerebral microenvironment, and disruption of this barrier after a stroke or trauma has potentially fatal consequences. Reactive oxygen species (ROS), which are formed during these cerebrovascular accidents, have a key role in this disruption. ROS are formed constitutively by mitochondria and also by the activation of cell receptors that transduce signals from inflammatory mediators, e.g., activated phospholipase A2 forms arachidonic acid that interacts with cyclooxygenase and lipoxygenase to generate ROS. Endothelial NADPH oxidase, activated by cytokines, also contributes to ROS. There is a surge in ROS following reperfusion after cerebral ischemia and the interaction of the signaling pathways plays a role in this. ROS are transiently generated during reperfusion following cerebrovascular ischaemia, which results in the initial disruption of the blood-brain barrier. This allows plasma proteins into the brain substance that leads to gliosis and subsequent destruction of brain tissue. We have investigated the source of this reperfusion injury-generated ROS in single venular capillaries on the cortical surface of the brain in young anaesthetized rats. The permeability to sulforhodamine dye (588 Da) of single venular capillaries of rats (anaesthetized with pentobarbitone: 60 mg.kg-1 ip, an overdose of which was administered to kill humanely) was measured by the occlusion technique. Cerebral ischaemia-reperfusion was instigated by infusing into the internal carotid artery 40 µm starch microspheres that subsequently dissolve due to the action of plasma amylase. This produced a blockage of the middle cerebral artery territory and we found that the permeability of previously tight venules increased with the duration of the ischaemia. This permeability increase was sensitive to blocking the bradykinin B2 receptor, but was an order of magnitude greater than the maximum permeability response to acutely applied bradykinin. Acutely applied bradykinin itself results in a free radical scavenger sensitive and calcium entry dependent permeability response. The source of this ROS was shown to be both cyclooxygenase and lipoxygenase activated by arachidonic acid released by phospholipase A2 activated in response to the applied bradykinin. Prolonged application of bradykinin did not itself increase the response, but permeability did increase after 30 min, and this was blocked by inhibiting the interleukin-1 receptor. We also found that interleukin-1β (IL-1β) application before applying bradykinin resulted in a large potentiation of the response. Furthermore, the early component of the potentiated response was shown to be sensitive to inhibiting NOX-2 assembly (Fig. 1). The vascular permeability response to the starch microsphere injection was also measured in the whole of the affected hemisphere by using escape of horseradish peroxidase (HRP) into the tissue. 50 µm thick sections were incubated with diaminobenzidine (DAB) and the rate of development of the reaction product was used to calculate the concentration of HRP in the tissue by comparison with brains soaked in HRP standard solutions. The PS product was calculated after 30 minutes reperfusion from the time averaged plasma HRP concentration according to the Crone-Renkin equation. PS increase depended on the duration of the blockage of the circulation and was greatest on the pial surface and radial veins draining the cortex. These experiments confirmed that the permeability increase on reperfusion lasted for less than 1h, and could be much reduced by preventing the formation of the hydroxyl radical by treating the animal with desferrioxamine which chelates the ferrous ion, so preventing the Fenton reaction.