High-molecular-weight kininogen (HK) is an abundant plasma protein that plays a central role in activation of the kallikrein-kinin system. Cleavage of HK by plasma kallikrein results in release of the nonapeptide bradykinin (BK), leaving behind cleaved high-molecular-weight kininogen (HKa). Previous studies have demonstrated that HKa induces apoptosis of proliferating endothelial cells and inhibits angiogenesis in vivo, activities mediated primarily through its domain 5. However, the mechanisms by which these effects occur are not well understood. Here, we demonstrate that HKa induces apoptosis of endothelial cells cultured on gelatin, vitronectin, fibronectin, or laminin but not collagen type I or IV. The ability of HKa to induce endothelial-cell apoptosis is dependent on the generation of intracellular reactive oxygen species and associated with depletion of glutathione and peroxidation of endothelial-cell lipids, effects that occur only in cells cultured on matrix proteins permissive for HKa-induced apoptosis. Finally, the ability of HKa to induce endothelial-cell apoptosis is blocked by the addition of reduced glutathione or N-acetylcysteine. These studies demonstrate a unique role for oxidant stress in mediating the activity of an antiangiogenic polypeptide and highlight the importance of the extracellular matrix in regulating endothelial-cell survival.