The microvascular brain endothelial cells that form the blood-brain barrier (BBB) are exposed to oxidative stress during hypoxia-reoxygenation (H/R) such as occurs in ischemic stroke. H/R triggers signaling cascades that may affect various features of the BBB including upregulation of the multi-drug transporter protein, P-glycoprotein (Pgp) [1, 2]. This could have important implications for drug delivery to the brain following ischemic stroke. In the present study, the immortalised rat brain endothelial cell line, GPNT [3], was used as an in vitro system to examine the effects of H/R on Pgp expression and to investigate the role of reactive oxygen species (ROS) and nitric oxide (NO) in the signaling cascades involved in these effects. GPNT cells were cultured until 70% confluent and harvested into 0.1% SDS either 24h after application of 200µM hydrogen peroxide (H₂O₂) or 24h after reoxygenation, following 6h hypoxia (H/R). In each case, reduced serum medium (RSM) was used. Hypoxia was achieved by placing cells in deoxygenated RSM and maintaining them at 1% O₂ in N₂ with 6% CO₂. Pgp protein levels were assessed via Western blot analyses. Cells were also analysed for production of ROS or NO within 1h after application of H₂O₂ or after H/R. This was done via flow cytometry using the ROS-sensitive dye, 2′,7′-dichlorofluorescin diacetate (DCF-DA) (loaded at 10µM for 10min) or the NO-sensitive dye 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM-DA) (loaded at 5µM for up to 1h). On exposure to 200µM H₂O₂, intracellular ROS levels increased, showing DCF fluorescence of 339 ± 67% of the untreated control after 20 min (n=4, p<0.05, paired t test). The upregulation of Pgp protein seen after exposure to H₂O₂ was abolished following pre-treatment with 6000U/ml PEG-catalase (n=3). The NO donor, SNP (1-2mM) also increased Pgp protein levels (n=3). After H/R, there was a small increase in intracellular ROS levels, reaching a maximum 10min after reoxygenation. Intracellular NO levels were increased 1h after reoxygenation, being 243 ± 75% of untreated controls (n=4, p<0.05, paired t test). The increases in Pgp protein levels seen after H/R were partially inhibited by either PEG-catalase or by the nitric oxide synthase inhibitor, L-NAME (1-2mM) (n=3). These results suggest that generation of both H₂O₂ and NO are involved in the increased expression of Pgp in rat brain endothelial cells following H/R and add to our understanding of the mechanisms involved in the changes to the BBB that accompany stroke. After stroke, BBB integrity becomes compromised. Thus upregulation of Pgp may provide compensatory means to protect the brain though it may also lead to decreased drug access [4].