Prekallikrein (PK) activation on human umbilical endothelial cells (HUVEC) presumably leads to bradykinin liberation. On HUVEC, PK activation requires the presence of cell-bound high-molecular-weight kininogen (HK) and Zn²⁺. We examined the Zn²⁺ requirement for HK binding to and the consequences of PK activation on endothelial cells. Optimal HK binding (14 pmol/10⁶ HUVEC) is seen with no added Zn²⁺ in HEPES-Tyrode buffer containing gelatin versus 16–32 μM added Zn²⁺ in the same buffer containing bovine serum albumin. The affinity and number of HK binding sites on HUVEC are a dissociation constant of 9.6 ± 1.8 nM and a maximal binding of 1.08 ± 0.26 × 10⁷ sites/cell (means ± SD). PK is activated to kallikrein by an antipain-sensitive mechanism in the presence of HK and Zn²⁺on HUVEC, human microvascular endothelial cells, umbilical artery smooth muscle cells, and bovine pulmonary artery endothelial cells. Simultaneous with kallikrein formation, bradykinin (5.0 or 10.3 pmol/10⁶ HUVEC in the absence or presence of lisinopril, respectively) is liberated from cell-bound HK. Liberated bradykinin stimulates the endothelial cell bradykinin B2 receptor to form nitric oxide. Assembly and activation of PK on endothelial cells modulates their physiological activities.