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Phosphorylated kininogen and some of its fragments containing serine phosphorylated bradykinin ([pS6]-Bk) were identified in human serum and plasma by a phosphoproteomic approach. We report the kininogenase ability of human tissue and plasma kallikreins and tryptase to generate [pS6]-Bk or Lys-[pS6]-Bk having as substrate the synthetic human kininogen fluorescent fragment Abz-MISLMKRPPGF[pS386]PFRSSRI-NH2. The pharmacological assays of [pS6]-Bk showed it as a full B2 bradykinin receptor agonist in smooth muscle, it produces a portal liver hypertensive response in rat and mouse paw edema that lasts longer than Bk. The rat hypotensive response to infusions of Bk is greater than that of [pS6]Bk, both if injected through femoral vein or aorta. [pS6]-Bk was more resistant than Bk to kininase digestion performed with angiotensin converting enzyme, neprilysin, thimet oligopeptidase, aminopeptidase P and carboxypeptidase M. 1H-NMR experiments indicated that [pS6]-Bk has lower flexibility, with the pS6-P7 bond restricted to the trans conformation, and can explain [pS6]-Bk resistance to hydrolysis. In conclusion, [pS6]-Bk presenting lower activity than Bk, with longer lasting effects and being slowly released by kininogenases from synthetic Abz-MISLMKRPPGF[pS386]PFRSSRI-NH2, suggests that phosphorylation of the kininogens can be an efficient kallikrein-kinin system regulator.