The soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) is a polypeptide secreted by endothelial cells that contains six out of the seven extracellular Ig-like domains of the transmembrane VEGFR-1 and a unique sequence of 30 amino acids. The biological role of sVEGFR-1 has not been clarified yet, even though it is considered a negative modulator of VEGF and PlGF activity, blocking the interaction of these growth factors with their membrane receptors. We have recently demonstrated that endothelial cells adhere on sVEGFR-1 and migrate in response to the polypeptide through a mechanism that involves the integrin alpha5beta1. Moreover, endothelial cells deposit the sVEGFR-1 into the extracellular matrix. These data suggest a role for sVEGFR-1 in processes of primary importance during angiogenesis, such as migration and interaction of endothelial cells with the extracellular matrix. To test this hypothesis, we designed a series of VEGFR-1 peptides, potentially able to block the sVEGFR-1/alpha5beta1 integrin interaction. Based on the structural analysis of VEGFR-1 second Ig-like domain, a set of eight peptides was synthesised. Peptides were tested for the ability to inhibit endothelial cell adhesion on sVEGFR-1 or fibronectin. Peptide A4 reduced cell adhesion on sVEGFR-1 and fibronectin by 50% and 30%, respectively. We next evaluated the effects of peptide A4 on endothelial cell migration induced by fibronectin, sVEGFR-1, VEGF or PlGF. Treatment with peptide A4 resulted in 30% inhibition of basal migration (without stimulus) and abrogation of the response to either sVEGFR-1 or PlGF, whereas it did not affect migration induced by fibronectin or VEGF. A control peptide, containing the amino acids of peptide A4 in a scrambled sequence, did not have any effect on endothelial cell migration. To further define the region of peptide A4 responsible of its inhibitory activity, a second set of peptides corresponding to a part of peptide A4 sequence was tested. Peptide B3 resulted to be the most efficient in inhibiting endothelial cell migration. In conclusion, peptide B3 behaves as a selective inhibitor of endothelial cell migration induced by sVEGFR-1 or PlGF. As cell migration is a fundamental step in the process of angiogenesis, peptide B3 could represent a leading molecule for the design of new drugs to be used in the treatment of cancer or other angiogenesis-related diseases. Supported by the Italian Ministry of Health and Compagnia di San Paolo.