The blood-brain barrier (BBB) secretes brain interstitial fluid, which requires regulated transport of ions such as Na⁺, K⁺, H⁺, Cl^– and HCO₃^–. The BBB is compromised in pathological conditions associated with metabolic insult, including stroke. The aim of this study was to investigate the effects of a metabolic insult on intracellular pH (pH_i) and Na⁺/H⁺ exchange activity in primary cultures of rat brain capillary endothelial cells (RBECs). RBECs were grown from microvessels isolated from brains of rats killed in accordance with U.K. legislation. To measure pH_i, cells were plated on glass coverslips, loaded with the pH-sensitive fluorescent dye BCECF and their fluorescence measured in a fluorimeter. The RBECs were bathed in a nominally CO₂/HCO₃^– free Hepes buffered solution, pH 7.4, containing 10mM glucose. Na⁺/H⁺ exchange activity was measured as the Na⁺-driven pH_i recovery from an intracellular acid load induced by an NH₄⁺ pulse (200s exposure to 20 mM). The rate of recovery after the NH₄⁺ was removed was slowed (76±4.6 per cent, mean±SEM, n=11) by EIPA (5-(N-ethyl-N-isopropyl) amiloride, 5 mM) suggesting recovery depends primarily on a Na⁺/H⁺ exchanger, NHE. EIPA without an NH₄⁺ pulse induced a progressive acidification, 0.22±0.04 pH units in 400s (n=3). 2-DG (2-deoxy-D-glucose, 10 mM in glucose-free solution), which inhibits glycolysis, induced after a short lag a rapid decrease in pH_i (t1/2 < 100s) to a level 0.65±0.09 (n=4) pH units below the initial value. The lack of recovery suggests that the NHE was inhibited, but the speed and extent of the decrease indicates an additional effect. The inhibition of the NHE was examined further using the larger acid shift produced by an NH₄⁺ pulse. Recovery was slowed from 0.0071±0.0011 pH units s^-1 (n=12) to 0.0010±0.0004 pH units s^-1 (n=9, p<0.001, unpaired t-test) when 2-DG was present throughout the experiment or 0.0013±0.0006 pH units s^-1 (n=13, p<0.001) when it was present starting 200 s before the NH₄⁺ pulse. 2-DG is expected to lead to a run-down of ATP levels, which would inhibit NHE [1], and would release H⁺, which might account for the decrease in pH_i. However, this explanation is unlikely. Removal of glucose, addition of iodoacetate (2.5mM), or addition of 10μM azide had no significant effects on pH_i over 800 s or on the recovery from an acid load. Furthermore a preliminary experiment has failed to detect the rapid decrease in ATP that would be required to explain the observed changes in pH_i. These results therefore suggest that 2-DG has effects other than via inhibition of glycolysis.