Investigations were performed to define the factor XII (FXII) binding site(s) on cultured endothelial cells (HUVECs). Biotin- or fluorescein isothiocyanate (FITC)–FXII in the presence of 10 μM Zn²⁺ specifically binds to HUVEC monolayers or cells in suspension. Collagen-stimulated platelets release sufficient Zn²⁺ to support FXII binding. On laser scanning confocal microscopy or electron microscopy, FITC-FXII or Nanogold-labeled FXII, respectively, specifically bind to HUVECs. Antibodies to gC1qR, urokinase plasminogen activator receptor (uPAR) and, to a lesser extent, cytokeratin 1 (CK1) block FXII binding to HUVECs as determined by flow cytometry and soluble or solid phase binding assays. FITC-FXII on endothelial cells colocalizes with gC1qR, uPAR and, to a lesser extent, CK1 antigen. Combined recombinant soluble uPAR and CK1 inhibit 80% FITC-FXII binding to HUVECs. Peptide Y(39)HKCTHKGR(47) (YHK9) from the N-terminal region of FXII and peptide H(479)KHGHGHGKHKNKGKKNGKH(498) from HK's domain 5 cell-binding site block FITC-FXII binding to HUVECs. Peptide YHK9 also inhibits FXIIa's activation of prekallikrein and FXI on HUVECs. These combined investigations indicate that FXII through a region on its fibronectin type II domain binds to the same multiprotein receptor complex that comprises the HK binding site of HUVECs. However, plasma concentrations of HK and vitronectin inhibit FXII binding to HUVECs 100% and 50%, respectively, and plasma albumin and other proteins prevent a sufficient level of free Zn²⁺ to be available to support FXII binding to HUVECs. Thus, physiologic FXII expression on HUVECs is secondary to HK binding and highly restricted in its ability to initiate prekallikrein or FXI activation.