The vascular endothelial (VE) receptor protein tyrosine phosphatase (VE-PTP) associates with VE-cadherin and supports endothelial cell contact integrity. This complex is rapidly dissociated by adhesion of leukocytes to endothelial cells or by vascular endothelial growth factor. We have shown recently that this dissociation is indeed required for the opening of endothelial cell contacts during leukocyte extravasation in vivo. The leukocyte receptor and signaling mechanism that stimulates VE-cadherin/VE-PTP dissociation are unknown. Here, we identify vascular cell adhesion molecule 1 as the relevant receptor for lymphocytes in this process. As signaling steps downstream of this receptor, we determined the activation of Rac1, the generation of reactive oxygen species by nicotinamide adenine dinucleotide phosphate oxidase and the activation of the redox-sensitive tyrosine kinase Pyk2 as essential for VE-cadherin/VE-PTP dissociation. These signaling steps are also required for the dissociation induced by VE growth factor. Searching for the molecular mechanism of complex dissociation, we found that a model substrate of VE-PTP represented by a tyrosine-phosphorylated peptide of Tie-2 dissociates VE-PTP from VE-cadherin when introduced with the help of a Tat peptide. We suggest that lymphocyte binding to vascular cell adhesion molecule 1 triggers a signaling process that enables a VE-PTP substrate to dissociate VE-PTP from VE-cadherin, thereby facilitating efficient transmigration.