The tight junction has been characterized as a domain of fusion of the exoplasmic leaflets of the lipid bilayers from adjacent epithelial cells. Approximating membranes to within fusion distance is a thermodynamically unfavorable process and requires the participation of membrane-bridging or -fusion proteins. No known tight junction protein exhibits such activities. Annexin A2 (A2), in particular its heterotetramer (A2t), is known to form junctions between lipid bilayer structures through molecular bridging of their external leaflets. We demonstrate abundant A2 expression in MDCK II monolayers by two-D gel electrophoresis. Confocal immunofluorescence microscopic analysis suggests the bulk of A2 is located along the apical and lateral plasma membrane in its tetrameric configuration, consisting of two A2 and two p11 (an 11 kDa calmodulinrelated protein, S100A10) subunits. Immunocytochemistry and ultrastructural immunogold labeling demonstrate colocalization of the A2 subunit with bona fide tight junction proteins, ZO-1, occludin and claudin-1, at cell-cell contacts. Extracellular addition of a synthetic peptide, targeted to disrupt the binding between A2 and p11, completely aborts tight junction assembly in calcium chelation studies. We propose A2t as a member of a new class of tight junction protein responsible for the long-observed convergence of adjacent exoplasmic lipid leaflets in tight junction assembly.