Tumor metastasis involves many stage-specific adhesive interactions. The expression of several cell adhesion molecules, notably the integrin α_vβ₃, has been associated with the metastatic potential of tumor cells. In this study, we used a novel in vitro assay to examine the role of α_vβ₃ in the transmigration of melanoma cells through a monolayer of human lung microvascular endothelial cells. Confocal microscopy revealed the presence of the integrin α_vβ₃ on melanoma membrane protrusions and pseudopods penetrating the endothelial junction. α_vβ₃ was also enriched in heterotypic contacts between endothelial cells and melanoma cells. Transendothelial migration of melanoma cells was inhibited by either a cyclic Arg-Gly-Asp peptide or the anti-α_vβ₃monoclonal antibody LM609. Although both platelet endothelial cell adhesion molecule-1 and L1 are known to bind integrin α_vβ₃, only L1 serves as a potential ligand for α_vβ₃ during melanoma transendothelial migration. Also, polyclonal antibodies against L1 partially inhibited the transendothelial migration of melanoma cells. However, addition of both L1 and α_vβ₃ antibodies did not show additive effects, suggesting that they are components of the same adhesion system. Together, the data suggest that interactions between the integrin α_vβ₃ on melanoma cells and L1 on endothelial cells play an important role in the transendothelial migration of melanoma cells.