Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca²⁺ ([Ca²⁺]_i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca²⁺]_i store depletion, but not extracellular Ca²⁺ chelation, prevented [Ca²⁺]_i elevation in response to O₂^.− that was inositol 1,4,5-trisphosphate (InsP₃) dependent, and cells lacking the three InsP₃ receptor (InsP₃R) isoforms failed to display the [Ca²⁺]_i transient. Importantly, the O₂^.−-triggered Ca²⁺ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O₂^.− and could be prevented by [Ca²⁺]_i buffering. This study provides evidence that O₂^.− facilitates an InsP₃R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation.