We have previously demonstrated that plasmin acts as a potent proinflammatory activator of human peripheral monocytes. Here we identify the annexin A2 heterotetramer, composed of annexin A2 and S100A10, as a receptor for the plasmin-induced signaling in human monocytes. Monocytes express the annexin A2 heterotetramer on the cell surface as shown by flow cytometry, fluorescence microscopy, and coimmunoprecipitation of biotinylated cell surface proteins. Binding of plasmin to annexin A2 and S100A10 on monocytes was verified by biotin transfer from plasmin labeled with a trifunctional cross-linker. Antibodies directed against annexin A2 or S100A10 inhibited the chemotaxis elicited by plasmin, but not that induced by fMLP. Further, down-regulation of annexin A2 or S100A10 in monocytes by antisense oligodeoxynucleotides impaired the chemotactic response to plasmin, but not that to fMLP. Antisense oligodeoxynucleotides similarly decreased the TNF-alpha release by plasmin-stimulated, but not by LPS-stimulated, monocytes. At the molecular level, stimulation with plasmin, but not with catalytically inactivated plasmin, induced cleavage of annexin A2 and dissociation of the heterotetramer complex. Substitution of lysine to alanine in position 27 abolished the cleavage of recombinant annexin A2 in vitro. Together, these data identify the annexin A2 heterotetramer as a signaling receptor activated by plasmin via proteolysis.