How single-chain urokinase (ScuPA) mediates angiogenesis is incompletely understood. ScuPA (≥4 nM) induces phosphorylated (p)ERK1/2 (MAPK44 and MAPK42) and pAkt (Ser473) in umbilical vein and dermal microvascular endothelial cells. Activation of pERK1/2 by ScuPA is blocked by PD-98059 or U-0126, and pAkt (Ser473) activation is inhibited by wortmannin or LY-294002. ScuPA (32 nM) or protease-inhibited two-chain urokinase stimulates pERK1/2 to the same extent, indicating that signaling is not dependent on enzymatic activity. ScuPA induces pERK1/2, but not pAkt (Ser473), in SIN1−/−cells, indicating that the two pathways are not identical. Peptides from domain 2 of the urokinase plasminogen activator receptor (uPAR) or domain 5 of high-molecular-weight kininogen compete with ScuPA for the induction of pERK1/2 and pAkt (Ser473). A peptide of the integrin-binding site on uPAR, a β1-integrin peptide that binds uPAR, antibody 6S6 to β1-integrin, tyrosine kinase inhibitors AG-1478 or PP3, and small interfering RNA knockdown of VEFG receptor 2, but not HER1–HER4, blocked ScuPA-induced pERK1/2 and pAkt (Ser473). ScuPA-induced endothelial cell proliferation was blocked by inhibitors of pERK1/2 and pAkt (Ser473), antibody 6S6, and uPAR or kininogen peptides. ScuPA initiated aortic sprouts and Matrigel plug angiogenesis in normal, but not uPAR-deficient, mouse aortae or mice, respectively, but these were blocked by PD-98059, LY-294002, AG-1478, or cleaved high-molecular-weight kininogen. In summary, this investigation indicates a novel, a nonproteolytic signaling pathway initiated by zymogen ScuPA and mediated by domain 2 of uPAR, β1-integrins, and VEGF receptor 2 leading to angiogenesis. Kininogens or peptides from it downregulate this pathway.