We previously reported that the bindingof two-chain high molecular weight kininogen(HKa) to endothelial cells may occur through interactionswith endothelial urokinase receptors. Sincethe binding of urokinase to urokinase receptorsactivates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding onendothelial cell proliferation. Unexpectedly, HKainhibited proliferation in response to several growthfactors, with 50% inhibition caused by; 10nM HKa.This activity was Zn21 dependent and not shared byeither single-chain high molecular weight kininogen(HK) or low molecular weight kininogen. HKa selectivelyinhibited the proliferation of human umbilicalvein and dermal microvascular endothelial cells, butdid not affect that of umbilical vein or human aorticsmooth muscle cells, trophoblasts, fibroblasts, orcarcinoma cells. Inhibition of endothelial proliferationby HKa was associated with endothelial cellapoptosis and unaffected by antibodies that blockthe binding of HK or HKa to any of their knownendothelial receptors. Recombinant HK domain 5displayed activity similar to that of HKa. In vivo, HKainhibited neovascularization of subcutaneously implantedMatrigel plugs, as well as rat corneal angiogenesis.These results demonstrate that HKa is anovel inhibitor of angiogenesis, whose activity isdependent on the unique conformation of the twochainmolecule.