The purpose of the present study was to examine the effect of short duration H2O2 exposure on coronary artery endothelial cell [Ca2+](i) regulation. Freshly dispersed cells from porcine coronary artery were exposed to H2O2 (300 mu mol/L) for 3 min while monitoring [Ca2+](i) using fura-2 microfluorometry. H2O2 increased [Ca2+](i) from 0.86 +/-0.03 to 2.19 +/- 0.41 ratio units at 3 min of H2O2 (P < 0.05). Intracellular Ca2+ remained elevated 3 min following removal of H2O2, yet H2O2 had no effect on the subsequent [Ca2+](i) response to bradykinin (0.1 mu mol/L). The H2O2-induced [Ca2+](i) increase was completely abolished either by removal of extracellular Ca2+ or lowering extracellular Na+. Cells exposed to the Na+ ionophore, monensin, showed an increase in [Ca2+](i) with a time course similar to that seen with H2O2. Furthermore, H2O2-induced Ca2+ influx was not attenuated by either Ni2+ (300 mu mol/L) or econazole (10 mu mol/L), excluding Ca2+ influx via the agonist-sensitive pathway. Thus, in coronary arterial endothelial cells, H2O2 increases Ca2+ influx in an extracellular Na+-dependent manner via an agonist-insensitive pathway.