Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O₂^.−) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O₂^.−, pulmonary microvascular endothelial cells were preloaded with the O₂^.−-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O₂^.− resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O₂^.− triggered Ca²⁺ release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O₂^.− production and cellular apoptosis. These “signaling” effects of O₂^.− were prevented by DIDS treatment, by depletion of intracellular Ca²⁺ stores with thapsigargin and by chelation of intracellular Ca²⁺. This study demonstrates that O₂^.− flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca²⁺ release, which activates mitochondrial O₂^.− generation.