Thrombospondins are a family of extracellular matrix proteins expressed throughout the developing nervous system that promote neurite outgrowth in vitro and help mediate the migration of granule cells across the molecular layer in explants of neonatal cerebellum. The receptors mediating these interactions have not previously been identified. In this study, monoclonal antibodies raised to the integrin alpha 3 beta 1 heterodimer are shown to inhibit neurite outgrowth by rat sympathetic neurons on thrombospondin-1. Alpha 3 beta 1 is found to be expressed on the cell body, neurites, and growth cones of sympathetic neurons in vitro and on sympathetic axons passing through the thrombospondin-rich outer sheath of the superior cervical ganglion in vivo, consistent with its role in mediating axon outgrowth. A receptor-ligand binding assay is used to demonstrate the direct binding of immunopurified alpha 3 beta 1 to thrombospondin-1. These results demonstrate a direct interaction between the integrin alpha 3 beta 1 and thrombospondin-1, which mediates neurite outgrowth in vitro and is likely to mediate the same interactions in vivo.