Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.