The drug transporter, P-glycoprotein (P-gp) on brain microvessel endothelium, influences movement of lipophilic substances in and out of the brain. Pathways regulating P-gp expression, both basal and hydrogen peroxide (H2O2)-induced, are here examined in primary cultured rat brain endothelial cells. Activation of extracellular-signal regulated kinases (ERK1/2), protein kinase C (PKC), the p46 isoform of stress-activated protein kinase (SAPK) and its downstream transcription factor, c-Jun, occurred in a time- and concentration-dependent manner following exposure of cells to H2O2 with concomitant increases in P-gp expression. Blockade of ERK activation with U0126, of PKC with Gö6976 and of SAPK with SP600125 decreased basal P-gp but did not abolish the H2O2-induced increase. Blockade of Akt with PI3-kinase inhibitor, LY294002, lowered basal P-gp and prevented the H2O2-induced increase. Inhibition of nuclear factor-kappaB (NF-kappaB), either by blocking dissociation from its inhibitory factor, IkappaB, with MG132 or its nuclear translocation with SN50 enhanced basal P-gp, obscuring the H2O2-induced increase. H2O2 itself produced no detectable activation of IkappaB, but inhibited that induced by 5 ng/mL tumour necrosis factor-alpha (TNF-alpha). P-gp expression may involve positive inputs from ERK1/2, SAPK, Akt and PKC and inhibitory influences of NF-kappaB. By depressing NF-kappaB signalling, H2O2 may still augment P-gp expression when ERK1/2, PKC or SAPK are inhibited.