Na⁺/H⁺ exchange activity has been examined in endothelial cells isolated from porcine brain capillaries. Intracellular pH (pH_i) changes were monitored using a confocal laser scanning microscope and the pH-sensitive fluorescence indicator 2′,7′-bis-(2-car☐yethyl)-5,6-car☐yfluorescein (BCECF). Acid load of the brain capillary endothelial cells was performed with a NH₄Cl (20 mM) prepulse. In bicarbonate-free solutions pH_i recovered within 3 to 10 min. Removal of extracellular Na⁺ ions demonstrated that H⁺ extrusion after an acid load of the cells was Na⁺ dependent. The Na⁺/H⁺ exchange could be completely blocked by EIPA (5-(N-ethyl-N-isopropyl)amiloride) as well as by the novel inhibitor 3-methylsulfonyl-4-piperidinobenzoyl guanidine hydrochloride (HOE 694) in concentrations of 1 to 10 μM, respectively. EIPA and HOE 694 in a concentration of 0.1 μM caused a partial block of Na⁺/H⁺ exchange.