Endothelial cells play a crucial role in maintaining a nonthrombogenic blood-tissue interface and regulate thrombosis, thrombolysis, platelet adherence, vascular tone, blood flow and they are indispensable for body homeostasis. High molecular weight kininogen (HK) is a plasma protein that interacts with blood and vascular cells. The peptide sequence of HK may be divided in different domains and it may function as antithrombin, profibrinolytic and antiadhesive molecule and it is also a potent local regulator of blood pressure through bradykinin delivery. HK assembles to endothelial cells through a multiprotein complex, and glycosaminoglycans also participate in this process. The aim of this work was to study the interaction of HK on endothelial cell surface and the role of cysteine peptidases and glycosaminoglycans in this process. Our cell model was a well established endothelial cell line obtained from rabbit aorta (RAECs). HK binding to cell surface was analyzed in buffers containing gelatin or albumin and biotin-HK binding was dependent on Zn²⁺. In the presence of albumin and different temperatures HK bound to RAECs in concentration dependent manner. At 37°C the K_dapp was 6.09 ± 0.38 nM and the B_max was 0.64 ± 0.01 A_450nm, and at 4°C the EC₅₀ was 19.3 nM. In the presence of gelatin and at 37°C, the K_dapp was 4.61 ± 0.84 nM and the B_max was 0.34 ± 0.01 A_450nm. HK binding was 43% reversible by 20 fold molar excess of unlabelled HK, 30% reversible by 500 fold molar excess of BK and it was completely reversible by 500 fold molar excess of HKH20. Intact HK (140 kDa) was hydrolyzed after 3 h bound to RAEC surface and a 82 kDa fragment was detected which intensity increased over time up to 9 h; in the presence of E-64 or cathepsin B inhibitor HK hydrolysis was blocked and anti-cathepsin B did not block HK hydrolysis. HK colocalized with cathepsin B and heparan sulfate on cell surface and it was internalized. The data shows that HK bound to RAEC surface is internalized and probably the interaction with heparan sulfate contributes to this process. HK may be cleaved by a cysteine peptidase and the HK fragment could play role in pathophysiological processes. (Supported by FAPESP/CNPq/FADA-UNIFESP).