A kininogen binding protein(s), a putative receptor, was identified on endothelial cells. A 54-kDa protein was isolated by a biotin–high molecular mass kininogen (HK) affinity column that, on aminoterminal sequencing of tryptic digests, was identified as cytokeratin 1. Multiple antibodies directed to cytokeratin 1 reacted with a 54-kDa band on immunoblot of lysates of endothelial cells. On laser scanning confocal microscopy, cytokeratin 1 antigen was found on the surface of endothelial cells. Cytokeratin 1 antigen also was detected on endothelial cell membranes by flow cytometry. Moreover, an antipeptide antibody to a sequence unique to cytokeratin 1 also specifically bound to nonpermeabilized endothelial cells. Biotin–HK specifically bound to cytokeratin only in the presence of Zn2+, and cytokeratin blocked biotin–HK binding to endothelial cells. Further, HK and low molecular mass kininogen, but not factor XII, blocked biotin–HK binding to cytokeratin, and peptides of each cell binding region of HK on domains 3,4, and 5 blocked biotin–HK binding to cytokeratin. gC1qR and soluble urokinase-like plasminogen activator receptor also inhibited biotin–HK binding to cytokeratin. These investigations identify a new function for cytokeratin 1 as a kininogen binding protein. Cytokeratins, members of the family of intermediate filament proteins, may represent a new class of receptors.