The purpose of this study is to further define transport pathways for biological thiols by blood-brain barrier (BBB) endothelial cells, as a means of identifying endogenous cytoprotective mechanisms and potential therapeutic protocols for oxidative injury. Similar low-affininty, high-capacity passive carriers for glutathione (GSH) were observed at both the luminal (blood-facing) and abluminal (brain-facing) plasma membranes of BBB endothelial cells. These carriers are voltage dependent, favoring outward movement of intact peptide across both membrane domains, including efflux at the luminal plasmalemma where γ-glutamyl transpeptidase is located. Although present at both cell surfaces, the carriers are distributed unequally, with more appearing in the abluminal membrane. By contrast, high-affinity, low-capacity sodium-dependent GSH cotransport (Na-GSH) is observed only at the abluminal membrane, indicative of an inwardly directed active peptide carrier at the brain-facing plasma membrane. Treatment of cultured BBB endothelial cells with the GSH precursor γ-glutamyl-cysteine reduces cell damage under conditions simulating ischemia and reperfusion. These findings are consistent with the presence of (1) a typical γ-glutamyl cycle at the luminal membrane of BBB endothelial cells, (2) a significant efflux pathway at the abluminal membrane allowing passive movement of BBB GSH into brain extracellular fluid, (3) a Na-dependent, brain-to-blood pathway for transcellular transport of GSH, and (4) a mechanism for cytoprotection by γ-glutamyl cysteine, under conditions of ischemia and reperfusion.