The integrin alpha3beta1 plays important roles in development, angiogenesis, and the pathogenesis of cancer, suggesting potential therapeutic uses for antagonists of this receptor. Recently, an alpha3beta1 integrin-binding site was mapped to residues 190-201 (FQGVLQNVRFVF) of the N-terminal domain of the secreted protein thrombospondin-1 (TSP1). This sequence displays diverse biological activities in vitro and inhibits angiogenesis in vivo. Herein we describe the NMR solution conformation of this segment in both water and dodecylphosphocholine micelles. While essentially unstructured in water, a more well-defined conformation is populated in micelles, particularly in the C-terminal half of the peptide and correlated with increased biological activity of the micellar peptide. The data suggested that the residues that are critical for biological activity are contained in a structurally well-defined segment of the peptide. These data support the role of the NVR motif as a required element of full-length TSP1 for specific molecular recognition by the alpha3beta1 integrin.