Levels of multidrug efflux transporter P-glycoprotein (P-gp) on endothelial cells lining brain blood vessels are important for limiting access of many compounds to the brain. In vivo studies have indicated that ischaemia-reperfusion that generates reactive oxygen species also increases P-gp levels in brain endothelial cells. To investigate possible mechanisms, in vitro studies were performed on immortalised (GPNT) and primary rat brain endothelial cells. Exposure to hydrogen peroxide (200 microM) resulted in intracellular oxidative stress as detected from higher levels of dichlorofluorescein fluorescence and raised levels of P-gp protein, mdr1a and mdr1b transcripts and, in GPNT cells, increased mdr1a and mdr1b promoter activity. The P-gp protein increases were abolished by pre-treatment with polyethylene glycol-catalase and were curtailed by co-culture with primary rat astrocytes. Exposure of GPNT cells to 6 h hypoxia followed by 24 h reoxygenation produced less intracellular oxidative stress as judged from smaller increments in dichlorofluorescein fluorescence but still resulted in raised levels of P-gp protein, an effect partially abolished by pre-treatment with polyethylene glycol-catalase. However, transcript levels and promoter activities were not significantly increased. These data suggest that hydrogen peroxide contributes to P-gp up-regulation following hypoxia-reoxygenation but the underlying mechanisms of its actions differ from those occurring after direct hydrogen peroxide application.