ABSTRACT Vascular endothelial growth factor (VEGF) is a well‐established stimulator of vascular permeability and angiogenesis, whereas thrombospondin‐1 (TSP‐1) is a potent angiogenic inhibitor. In this study, we have found that the TSP‐1 receptors CD36 and ßl integrin associate with the VEGF receptor 2 (VEGFR2). The coclustering of receptors that regulate angiogenesis may provide the endothelial cell with a platform for integration of positive and negative signals in the plane of the membrane. Thus, this complex may represent a molecular switch that regulates angiogenesis and determines endothelial cell behavior. In this context, physiological levels of TSP‐1 appear to support VEGFR2 function on both the cellular and tissue level, because phosphorylation of VEGFR2 and vascular permeability in response to VEGF are decreased in TSP‐1‐null mice and isolated endothelial cells. A therapeutic agent based on the antiangiogenic domain of TSP‐1, designated 3TSR (for three TSP‐1 type 1 repeats), has significant antiangiogenic and antitumor efficacy. Systemic treatment of wild‐type mice with 3TSR significantly decreased VEGF‐induced permeability. Consistent with this result, VEGF‐stimulated phosphorylation of VEGFR2 was also significantly decreased in lung extracts from 3TSR‐treated mice. Moreover, 3TSR significantly decreased VEGF‐stimulated VEGFR2 phosphorylation in human dermal microvascular endothelial cells in culture. Taken together, the results indicate that TSP‐1 and 3TSR modulate the function of VEGFR2.—Zhang, X., Kazerounian, S., Duquette, M., Perruzzi, C., Nagy, J. A., Dvorak, H. J., Parangi, S., and Lawler, J. Thrombospondin‐1 modulates vascular endothelial growth factor activity at the receptor level. FASEB J . 23, 3368–3376 (2009). www.fasebj.org