The regulation of intracellular free Ca2+ concentration ([Ca2+](i)) was studied in cultured human aortic endothelial cells loaded with the fluorescent Ca2+ indicator fura-2. Histamine and ATP at concentrations of 10 mu M and higher produced a biphasic change in [Ca2+](i), which consisted of an initial transient elevation followed by a sustained elevation. Reduction of the extracellular Cl- concentration to 40 mM, or exposure to the Cl- channel antagonist N-phenylanthranilic acid selectively prevented the sustained response to histamine or ATP, but they did not affect the sustained response to the Ca2+ ionophore ionomycin. Elevation of extracellular Kf concentration to 90 mM had no influence on the sustained response to histamine or ATP. These results suggest that the sustained elevation of [Ca2+](i) in response to histamine and ATP is due to the Cl--sensitive entry of extracellular Ca2+ in cultured human aortic endothelial cells.