The junctional adhesion molecules (JAMs) have been recently described as interendothelial junctional molecules and as integrin ligands. Here we show that JAM-B and JAM-C undergo heterophilic interaction in cell-cell contacts and that JAM-C is recruited and stabilized in junctional complexes by JAM-B. In addition, soluble JAM-B dissociates soluble JAM-C homodimers to form JAM-B/JAM-C heterodimers. This suggests that the affinity of JAM-C monomers to form dimers is higher for JAM-B than for JAM-C. Using antibodies against JAM-C, the formation of JAM-B/JAM-C heterodimers can be abolished. This liberates JAM-C from its vascular binding partner JAM-B and makes it available on the apical side of vessels for interaction with its leukocyte counterreceptor α_Mβ₂ integrin. We demonstrate that the modulation of JAM-C localization in junctional complexes is a new regulatory mechanism for α_Mβ₂-dependent adhesion of leukocytes.